Mixed Cell Populations For Tissue Repair And Separation Technique For Cell Processing

ABSTRACT

The present invention provides a fluid exchange cell culture technique and tissue repair cells (TRCs) made by these methods, as well as methods using these cells. The method includes a new wash step which increases the tissue repair properties of the TRCs of the invention. This wash step allows for the production of TRC populations with greater tissue repair and anti-inflammatory capabilities. Embodiments of the present invention include a post-culture process for cultured cells that preferably includes the steps of: a wash process for removing unwanted residual culture components, a volume reduction process, and a harvesting process to remove cultured cells. Preferably, all these steps are performed within a aseptically closed cell culture chamber by implementing a separation method that minimizes mechanical disruption of the cells and is simple to automate. The harvested cells may then be concentrated to a final volume for the intended use. In such embodiments, the final composition is a substantially purified and concentrated cell mixture suspended in a physiologic solution suitable for immediate use in humans without further washing, volume reduction, or processing. Embodiments are also applicable to harvesting (and/or washing) particles within a liquid or solution within a chamber.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/983,008, filed Nov. 5, 2007, which claims the benefit of U.S. Ser.No. 60/856,504, filed Nov. 3, 2006 and U.S. Ser. No. 60/932,702, filed,Jun. 1, 2007, the contents of which are each herein incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions of mixed cell populations,their subsequent use in vivo for tissue repair and processes, devices,and systems for the preparation of the mixed cell populations. Theprocesses of the invention are also applicable at separating any type ofcell (adherent, non-adherent or a mixture thereof) or small particles(e.g., cell sized) from a containing liquid or solution.

BACKGROUND OF THE INVENTION

Regenerative medicine harnesses, in a clinically targeted manner, theability of regenerative cells, e.g., stem cells and/or progenitor cells(i.e., the unspecialized master cells of the body), to renew themselvesindefinitely and develop into mature specialized cells. Stem cells arefound in embryos during early stages of development, in fetal tissue andin some adult organs and tissue. Embryonic stem cells (hereinafterreferred to as “ESCs”) are known to become many if not all of the celland tissue types of the body. ESCs not only contain all the geneticinformation of the individual but also contain the nascent capacity tobecome any of the 200+ cells and tissues of the body. Thus, these cellshave tremendous potential for regenerative medicine. For example, ESCscan be grown into specific tissues such as heart, lung or kidney whichcould then be used to repair damaged and diseased organs. However, ESCderived tissues have clinical limitations. Since ESCs are necessarilyderived from another individual, i.e., an embryo, there is a risk thatthe recipient's immune system will reject the new biological material.Although immunosuppressive drugs to prevent such rejection areavailable, such drugs are also known to block desirable immune responsessuch as those against bacterial infections and viruses.

Moreover, the ethical debate over the source of ESCs, i.e., embryos, iswell-chronicled and presents an additional and, perhaps, insurmountableobstacle for the foreseeable future.

Adult stem cells (hereinafter interchangeably referred to as “ASCs”)represent an alternative to the use of ESCs. ASCs reside quietly in manynon-embryonic tissues, presumably waiting to respond to trauma or otherdestructive disease processes so that they can heal the injured tissue.Notably, emerging scientific evidence indicates that each individualcarries a pool of ASCs that may share with ESCs the ability to becomemany if not all types of cells and tissues. Thus, ASCs, like ESCs, havetremendous potential for clinical applications of regenerative medicine.

ASC populations have been shown to be present in one or more of bonemarrow, skin, muscle, liver and brain. However, the frequency of ASCs inthese tissues is low. For example, mesenchymal stem cell frequency inbone marrow is estimated at between 1 in 100,000 and 1 in 1,000,000nucleated cells Thus, any proposed clinical application of ASCs fromsuch tissues requires increasing cell number, purity, and maturity byprocesses of cell purification and cell culture.

Although cell culture steps may provide increased cell number, purity,and maturity, they do so at a cost. This cost can include one or more ofthe following technical difficulties: loss of cell function due to cellaging, loss of potentially useful cell populations, delays in potentialapplication of cells to patients, increased monetary cost, increasedrisk of contamination of cells with environmental microorganisms duringculture, and the need for further post-culture processing to depleteculture materials contained with the harvested cells.

More specifically, all final cell products must conform with rigidrequirements imposed by the Federal Drug Administration (FDA). The FDArequires that all final cell products must minimize “extraneous”proteins known to be capable of producing allergenic effects in humansubjects as well as minimize contamination risks. Moreover, the FDAexpects a minimum cell viability of 70%, and any process shouldconsistently exceed this minimum requirement.

While there are existing methods and apparatus for separating cells fromunwanted dissolved culture components and a variety of apparatuscurrently in clinical use, such methods and apparatus suffers from asignificant problem—cellular damage caused by mechanical forces appliedduring the separation process, exhibited, for instance, by a reductionin viability and biological function of the cells and an increase infree cellular DNA and debris. Furthermore, significant loss of cells canoccur due to the inability to both transfer all the cells into theseparation apparatus as well as extract all the cells from theapparatus. In addition, for mixed cell populations, these methods andapparatus can cause a shift in cell profile due to the preferential lossof larger, more fragile subpopulations.

Thus, there is a need in the field of cell therapy, such as tissuerepair, tissue regeneration, and tissue engineering, for cellcompositions that are ready for direct patient administration withsubstantially high viability and functionality, and with substantialdepletion of materials that were required for culture and harvest of thecells. Furthermore, there are needs for reliable processes and devicesto enable production of these compositions that are suitable forclinical implementation and large-scale commercialization of thesecompositions as cell therapy products.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for tissue repair. Thecomposition are useful for treating a variety of diseases and disorderssuch as ischemic conditions (e.g., limb ischemia, congestive heartfailure, cardiac ischemia, kidney ischemia and ESRD, stroke, andischemia of the eye), conditions requiring organ or tissue regeneration(e.g., regeneration of liver, pancreas, lung, salivary gland, bloodvessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney,muscle, cardiac muscle, nerve, and limb), inflammatory diseases (e.g.,heart disease, diabetes, spinal cord injury, rheumatoid arthritis,osteo-arthritis, inflammation due to hip replacement or revision,Crohn's disease, and graft versus host disease) and auto-immune diseases(e.g., type 1 diabetes, psoriasis, systemic lupus, and multiplesclerosis).

In one aspect the invention provides an isolated cell composition fortissue repair containing a mixed population of cells. The cells are in apharmaceutical-grade electrolyte solution suitable for humanadministration. The cells are derived from mononuclear cells. Forexample, the cells are derived from bone marrow, peripheral blood,umbilical cord blood or fetal liver. The cells are of hematopoietic,mesenchymal and endothelial lineage. The viability of cells is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater. The total number ofviable cells in the composition is 35 million to 300 million and involume less than 25 ml, 20 ml, 15 ml, 10 ml, 7.5 ml, 5 ml or less. Atleast 5% of the viable cells in the composition are CD90⁺. For example,10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or more are CD90⁺. In someaspects at least 5%, 10%, 15%, 20%, 50% or more of the CD90⁺ co-expressCD15. Preferably, the cells are about 5-75% viable CD90⁺ with theremaining cells in the composition being CD45⁺. The CD45⁺ cells areCD14⁺, CD34⁺ or VEGFR1⁺.

The cells produce at least one, two, three four, five or more anti-inflammatory cytokines or angiogenic factors. Anti-inflammatorycytokines include for example interleukin-1 receptor antagonist,interleukin-6, TGF-β, interleukin-8, interleukin 10, or monocytechemoattractant protein-1. Angiogenic factors include for example,vascular endothelial growth factor, angiopoeitin 1, angiopoeitin 2 orhepatocyte growth factor. Additionally the cells produce less than 50pg/mL, 40 pg/mL, 30 pg/mL, 20 pg/mL, 10 pg/mL, 5 pg/mL, 2 pg/mL or 1pg/mL per 24 hour period per 10⁵ cells of one or more pro-inflammatorycytokines such as interleukin-1 alpha, interleukin-1 beta, interferongamma or interleukin-12. The cells also express indoleamine 2,3,dioxygenase, PD-L1 or both.

The composition is substantially free of components used during theproduction of the cell composition, e.g., cell culture components suchas bovine serum albumin, horse serum, fetal bovine serum, enzymaticallyactive harvest reagent (e.g., trypsin) and substantially free ofmycoplasm, endotoxin, and microbial contamination . Preferably, thecomposition contain 10, 5, 4, 3, 2, 1, 0.1, 0.05 or less μg/ml bovineserum albumin and 5, 4, 3, 2, 1, 0.1, 0.05 μg/ml enzymatically activeharvest reagent.

Optionally, the composition further contains a bio-compatible matrixsuch as for example, demineralized bone particles, mineralized boneparticles, synthetic ceramic of the calcium phosphate family (e.g.,alpha tri-calcium phosphates, beta tri-calcium phosphates andhydroxyapatites), collagens, polysaccharide-based materials (e.g.,hyaluronan and alginates), synthetic biodegradable polymeric materials(e.g., poly-lactides, poly-glycolides, poly-fumarates and poly-ethyleneglycol), and mixtures, combinations or blends thereof.

In another aspect the invention provides methods of modulating an immuneresponse, an inflammatory response or angiogenesis in a patient byadministering a cultured mixed cell composition to the patient whereinthe cultured cell composition produces at least one cytokine such asinterleukin-1 receptor antagonist, interleukin-6, interleukin-8,interleukin-10, vascular endothelial growth factor, monocytechemoattractant protein-1 angiopoeitin 1, angiopoeitin 2 and hepatocytegrowth factor. Optionally, the cell composition produces two, three,four, five or more cytokines. Preferably, the composition produces lessthan 10 ng/mL of interleukin-1 alpha, interferon gamma orinterleukin-12. For example, the cell composition contains 0.1%- 10%CD4⁺CD24⁺ T-cells, 1-50% CD45⁺CD14⁺ monocytes: and 5% -75% CD45-CD90⁺bone marrow stromal cells. Preferably, the cell composition is the cellcompositions described above.

In a further aspect the invention provides a method for processingcultured cells. The method produces a mixed cell population wherein morethat 5% of the cell population is CD90⁺. The method includes: providinga biochamber for culturing cells, providing a culture media forculturing cells within biochamber, inoculating the biochamber withcells. The cells are cultured and upon a predetermined time period ofculture, displacing the culture media from the biochamber with abiocompatible first rinse solution substantially replacing the firstrinse solution with a cell harvest enzyme solution and incubating thecontents of the biochamber for a predetermined period of time such thatduring incubation, the enzyme at least dissociates the cells from eachother and/or from the biochamber surface. The enzyme solution isreplaced with a second rinse solution to displace the enzyme solution.The chamber is substantially filled with the second rinse solution.Preferably, the second rinse solution is a solution capable of beinginjectable into a human. Optionally, the method further comprises on ormore addition steps including displacing a portion of the second rinsesolution with a gas to obtain a predetermined reduced liquid volume inthe chamber; agitating the chamber to bring settled cells intosuspension and draining the solution with the suspended cells into acell collection container. After draining the solution into a cellcollection container, additional amounts of the second solution areadded to the biochamber, and the biochamber is agitated to rinse outresidual cells.

Also included in the invention are the cultured cells and compositioncontaining the cultured cells produced by the methods of the invention.

In another aspect the invention provides a method for harvestingcultured cells. The method includes the steps of displacing culturemedia from a biochamber with a biocompatible first rinse solution;substantially replacing the first rinse solution with a cell harvestenzyme solution and incubating the contents of the biochamber for apredetermined period of time with the enzyme solution. Duringincubation, the enzyme at least dissociates the cells from each otherand/or from culture surface of the biochamber. The enzyme solution isdisplaced with a second rinse solution. The chamber is substantiallyfilled with the second rinse solution.

Optionally, the method further includes on or more of the followingsteps: displacing a portion of the second rinse solution with a gas toobtain a predetermined reduced liquid volume in the chamber; agitatingthe chamber to bring settled cells into suspension; draining thesolution with suspended cells into a cell collection container. Afterdraining the solution into the cell collection container, additionalamounts of the second solution are added to the biochamber, and thebiochamber is agitated to rinse out the residual cells.

The cells are derived from mononuclear, for example the mononuclearcells are bone marrow, peripheral blood, umbilical cord blood or fetalliver.

In yet a further aspect the invention provides methods for separatingmicro-particles from a containing liquid or solution provided in achamber having a predetermined volume and geometry by introducing asecond liquid/solution within the chamber to displace the firstcontaining liquid, wherein the geometry of the chamber enables theliquids to flow through the chamber according to a plug-flow and thesecond liquid substantially displaces the volume of the chamber at leastonce. Optionally a gas is introduced at a rate to establish a plug-flow,wherein the gas displaces a liquid/solution contained in the chamber toreduce liquid/solution volume and thereby concentrate the particleswithin the liquid/solution in the chamber. The method also includesagitating the chamber to bring settled particles into theliquid/solution contained in the chamber and draining the solution intoa collection container.

The flow rate for the introduction of solutions and/or gases added tothe biochamber for any of the described methods is between about 0.03 toabout 1.0 volume-exchanges/min. Preferably, the flow rate for theintroduction of solutions and/or gases added to the biochamber isbetween about 0.50 to about 0.75 volume exchanges/min. Optionally, theliquids/solutions or gases are introduced into the biochamber accordingto a radial plug flow.

The second liquid or subsequent liquid/solution is capable of beinginjectable into a human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood from the following detailed description when considered inconnection with the accompanying drawings in which like referencecharacters designate like or corresponding parts throughout the severalviews and wherein:

FIG. 1 is a diagram illustrating the major components of the cellproduction system according to the invention.

FIG. 2 is a schematic illustration of one embodiment of the overallsystem of FIG. 1.

FIG. 3 is a schematic diagram illustrating another embodiment of theoverall system of FIG. 1.

FIGS. 4A and 4B are schematic top and side views of an embodiment of acell cassette according to the invention.

FIG. 5 is an exploded view of the cell cassette of FIGS. 4A and 4B.

FIG. 6 is a schematic view showing fluid conduit routing in the cellcassette according to an embodiment of the invention.

FIG. 7 is a schematic sectional view of the biochamber portion of a cellcassette according an embodiment of the invention;

FIGS. 8A and 8B are top and sectional views of a biochamber coveraccording to an embodiment of the invention.

FIGS. 9A and 9B are top and sectional views of a biochamber cell beddisc according to an embodiment of the invention.

FIGS. 10A and 10B are top and sectional views of a biochamber baseaccording to embodiment of the invention.

FIG. 11 is a bar graph showing the ratio of Wash-Harvest/CYTOMATE® washresults for % cell viability post-wash, post-concentration, andpost-storage; % CD90, % CD14auto+, % VegfR1+, CFU-F and CFU-GMfrequency, and residual bovine serum albumin (BSA).

FIG. 12 is a bar graph showing the ratio of Wash-Harvest/CYTOMATE® washresults for total viable cells post wash, total viable cells finalproduct, total viable CD90⁺ cells, total viable CD14⁺ auto⁺ cells, totalviable VEGFR1⁺ cells, total CFU-Fs, and total CFU-GMs.

FIG. 13 is a bar graph showing the ratio of CFU-F frequency,(Wash-harvest)/(CYTOMATE® wash).

FIG. 14 is a bar graph showing CFU-Fs per dose of TRCs. Total post-washviability cell count measured by Nucleocounter, was used to calculateCFU-Fs/dose, except where not available and trypan blue data was used(samples 106-70 and 106-72). For each pair of bars for each sample, theleft bar shows results using the CYTOMATE® wash and the right bar showsresults using the wash-harvest.

FIG. 15 is a bar graph showing the ratio of CFU-GM frequency,(Wash-harvest)/(CYTOMATE® wash).

FIG. 16 is a bar graph showing CFU-GMs per dose of TRCs. Total post-washviability cell count measured by Nucleocounter, was used to calculateCFU-GMs/dose, except where not available and trypan blue data was used(samples 106-70 and 106-72). For each pair of bars for each sample, theleft bar shows results using the CYTOMATE® wash and the right bar showsresults using the wash-harvest.

FIG. 17 is a bar graph showing the total viability of TRCs afterdelivery through needles measured by Nucleocounter after undergoing thewash-harvest. The bar on the left represents the control, the middle barrepresents a 25 gauge needle and the right bar represents a 30 gaugeneedle for each experiment.

FIG. 18 is a bar graph showing the total viability of TRCs afterdelivery through needles measured by Nucleocounter after undergoing theCYTOMATE® wash. The bar on the left represents the control, the middlebar represents a 25 gauge needle and the right bar represents a 30 gaugeneedle for each experiment.

FIG. 19 is a bar graph showing CFU-Fs after 24 hour storage and needledelivery. For each pair of bars for each sample, the left bar showsresults using the CYTOMATE® wash and the right bar shows results usingthe wash-harvest.

FIG. 20 is a bar graph showing the normalized Wash-harvest/CYTOMATE®wash cytokine dose for a number of cytokines on TRCs.

FIG. 21 is a bar graph showing the osteogenic potential of CYTOMATE®wash TRCs and Wash Harvest TRCs.

FIG. 22A is a bar graph showing the amount of calcium produced per CD90⁺cell plated for TRCs and mesenchymal stem cells (MSCs).

FIG. 22B is a bar graph showing the amount of alkaline phosphataseproduced per CD90⁺ cell plated for TRCs and mesenchymal stem cells(MSCs).

FIG. 23A shows a flow cytometric analysis of TRCs produced using thewash-harvest stained for two-color analysis using irrelevantisotype-matched control monoclonal antibodies (mAbs) (IgG1, IgG2a).

FIG. 23B shows a flow cytometric analysis of TRCs produced using thewash-harvest stained with specific fluorochrome-conjugated anti-CD25plus anti-CD4 monoclonal antibodies (mAbs).

FIG. 23C is a bar graph showing the cytokine secretion profile ofT-cells within the TRC mixture after specific activation with ananti-CD3 monoclonal antibody (mAb) designated OKT3. This monoclonalantibody cross-links the CD3-T-cell receptor (TCR) cell surface complexthus triggering cytokine release by T-cells. Luminex® analysis was usedto evaluate IL-2, IFNγ and IL-10 release into the supernatant fluidcollected 48 hours after T-cell activation using OKT3 mAb.

FIG. 24 is a bar graph showing the relative amount of Indoleamine2,3-Dioxygenase (IDO) message expressed in IFNγ induced TRCs whendetermined by quantitative polymerase chain reaction (qPCR). The mean oftriplicate samples are shown for each determination.

FIG. 25 is a Western blot showing the expression of Indoleamine2,3-Dioxygenase protein in IFNγ induced TRCs.

FIG. 26 is a bar graph showing HGF production by TRCs.

FIG. 27 is a bar graph showing the percent of IDO positive cells in IFNγinduced TRCs as determined by flow cytometry.

FIG. 28 is a bar graph showing the percent of PDL1 positive cells inIFNγ induced TRCs as determined by flow cytometric analysis.

FIG. 29A is a graph showing ³H-thymidine incorporation in an allogeneicmixed leukocyte response (MLR) in the presence of allogeneic T-cells anddendritic cells as compared to TRCs.

FIG. 29B is a graph showing ³H-thymidine incorporation in an allogeneicmixed leukocyte response (MLR) in the presence of allogeneic T-cellsplus dendritic cells compared to TRCs.

FIG. 30 is a bar graph showing ³H-thymidine incorporation in anallogeneic mixed leukocyte response (MLR) in the presence of allogeneicT-cells together with increasing doses of TRCs without (uninduced) orwith (induced) exposure to IFNγ.

FIG. 31 shows X-rays of a patient who fell from a scaffold and wastreated with TRCs for fracture of both tibias.

FIG. 32A shows a photomicrograph histology slide of new bone in ahealing callus.

FIG. 32B shows a bright field photomicrograph histology slide of bloodvessels and new bone penetrating the allograft.

FIG. 32C shows a polarized light photomicrograph histology slide ofblood vessels and new bone penetrating the allograft.

FIG. 33A is a photograph of an implantable TRC/demineralized bone matrix(DBM) mixture that has been bound with autologous plasma.

FIG. 33B is a photomicrograph of a 24 hour live/dead stain of theTRC/DBM mixture at 4×.

FIG. 34A is a graph showing that TRCs in the RC/DBM allograft are viablepost mixing and proliferate over a two week period.

FIG. 34B is a photomicrograph of a 14 day live/dead stain of the TRC/DBMmixture at 4×.

FIG. 35 displays graphs showing that TRCs maintain secretion ofosteocalcin, IL-6, osteoprotegrin and VEGF throughout two weeks ofculture in a TRC/DBM mixture.

FIG. 36 are photographs showing the Toe of 69 year old male patienttreated with TRCs. Before treatment (left) a non-healing wound wasobserved. 44 weeks after treatment (right) complete healing wasobserved. The patient suffered from numerous co-morbidities includingcoronary heart disease, chronic heart failure, hypertension andhyperlipidemia.

FIG. 37 are photographs showing MR-angiography of limbs of 69 year oldmale patient treated with TRCs. This patient received TRC injections inthe right limb. Before treatment (left panel) very littlecollateralization is observed. 48 weeks after treatment (right panel)significantly more collaterals can be observed in the treated limb. Thepatient suffered from numerous co-morbidities including coronary heartdisease, hypertension and hyperlipidemia.

FIG. 38 is a bar chart showing the increase or decrease of certain celltypes in TRCs compared to BM MNCs.

FIG. 39 is a illustration showing the frequency of hematopoietic andmesenchymal elements in BM MNCs and TRCs.

FIG. 40 is a bar chart showing that the cytokine production profiles aresignificantly different between BM MNCs and TRCs from the same donor.

FIG. 41A-C is a series of bar charts showing the frequency of CD90 andCFU-f in MSC and TRC cultures. MSC and TRC were generated in theautomated bioreactor system as described in Materials and Methods. Thefrequency of CD90 and CFU-f in the output culture are shown in A. and B.respectively. The CFU-f frequency was then calculated based on thenumber of CD90 cells in each product. Results are shown in C. Dark barsrepresent TRC cultures and open bars represent MSC cultures. Twoindependent normal donors are shown.

FIG. 42 is a line graph showing comparison of bone formation in vivo inan ectopic mouse model. Bone scores were determined for each loadingcell density from MSC and TRC cultures. the graph shows the calculatedloading dose of CD90+ cells from each culture. The results presented arerepresentative experiment from one normal donor. In this experiment, MRCwere 68% CD90⁺ and TRCs were 22% cCD0⁺.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of compositions andmethods of producing cells for cell therapy. The compositions are amixed population of cells that are enhanced in stem and progenitor cellsthat are uniquely suited to human administration for tissue repair,tissue regeneration, and tissue engineering. These cells are referred toherein as “Tissue Repair Cells” or “TRCs”

Accordingly, in one aspect the invention provides a compositioncontaining a mixed population of cells of hematopoietic, mesenchymal andendothelial lineage. The composition is suitable for administration to ahuman for therapeutic use. TRCs are produced from an in vitro cultureprocess. Once the culture process is completed, culture components (e.g.culture medium, enzyme used for detachment and harvest of the cells)must be separated from the cells before they can be safely administeredto a subject in need of tissue regeneration. This separation isconventionally performed in a post-culture cell washing step. However, asignificant problem associated with this step is cellular damage causedby mechanical forces applied during these processes, exhibited, forinstance, by a reduction in viability and biological function of thecells and an increase in free cellular DNA and debris. This loss ofviability and function has not only immediate impact on the cellproduct, but also greatly impacts the shelf-life and cryopreservationpotential of the cells. Additionally, significant loss of cells canoccur due to the inability to both transfer all the cells into thewashing apparatus as well as extract all the cells from the apparatus.

Accordingly, in another aspect the invention provides a cell washingprocedure. The cell washing techniques of the instant invention, asdescribed in the Methods of Production of TRCs below, surprisinglygreatly enhanced cell viability and yield compared to currentpost-culture wash procedures while providing cell compositions withresidual levels of culture components that are sufficiently low for safeadministration of the cells to a patient.

Tissue Repair Cells (TRCs)

Tissue Repair Cells (TRCs) provide a cellular and molecular compositionwith high functionality for repair of injured tissues. Additionally, theTRCs have been shown to have anti-inflammatory effects. TRCs contain amixture of cells of hematopoietic, mesenchymal and endothelial celllineage produced from mononuclear cells. The mononuclear cells areisolated from adult, juvenile, fetal or embryonic tissues. For example,the mononuclear cells are derived from bone marrow, peripheral blood,umbilical cord blood or fetal liver tissue. TRCs are produced frommononuclear cells for example by an in vitro culture process whichresults in a unique cell composition having both phenotypic andfunctional differences compared to the mononuclear cell population thatwas used as the starting material. Additionally, the TRCs of the instantinvention have both high viability and low residual levels of componentsused during their production.

The viability of the TRC's is at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95% or more. Viability is measured by methods known in the art suchas trypan blue exclusion. This enhanced viability makes the TRCpopulation more effective in tissue repair, as well as enhances theshelf-life and cryopreservation potential of the final cell product.

By components used during production is meant but not limited to culturemedia components such as horse serum, fetal bovine serum and enzymesolutions for cell harvest. Enzyme solutions include trypsins(animal-derived, microbial-derived, or recombinant), variouscollagenases, alternative microbial-derived enzymes, dissociationagents, general proteases, or mixtures of these. Removal of thesecomponents provide for safe administration of TRC to a subject in needthereof.

Preferably, the TRC compositions of the invention contain less than 10,5, 4, 3, 2, 1 μg/ml bovine serum albumin; less than 5, 4, 3, 2, 1, 0.9,0.8, 0.7, 0.6, 0.5 μg/ml harvest enzymes (as determined by enzymaticactivity) and are substantially free of mycoplasm, endotoxin andmicrobial (e.g., aerobic, anaerobic and fungi) contamination.

By substantially free of endotoxin is meant that there is less endotoxinper dose of TRCs than is allowed by the FDA for a biologic, which is atotal endotoxin of 5 EU/kg body weight per day, which for an average 70kg person is 350 EU per total dose of TRCs.

By substantially free for mycoplasma and microbial contamination ismeant as negative readings for the generally accepted tests know tothose skilled in the art. For example, mycoplasm contamination isdetermined by subculturing a TRC product sample in broth medium anddistributed over agar plates on day 1, 3, 7, and 14 at 37° C. withappropriate positive and negative controls. The product sampleappearance is compared microscopically, at 100×, to that of the positiveand negative control. Additionally, inoculation of an indicator cellculture is incubated for 3 and 5 days and examined at 600× for thepresence of mycoplasmas by epifluorescence microscopy using aDNA-binding fluorochrome. The product is considered satisfactory if theagar and/or the broth media procedure and the indicator cell cultureprocedure show no evidence of mycoplasma contamination.

The sterility test to establish that the product is free of microbialcontamination is based on the U.S. Pharmacopedia Direct Transfer Method.This procedure requires that a pre-harvest medium effluent and apre-concentrated sample be inoculated into a tube containing tryptic soybroth media and fluid thioglycollate media. These tubes are observedperiodically for a cloudy appearance (turpidity) for a 14 dayincubation. A cloudy appearance on any day in either medium indicatecontamination, with a clear appearance (no growth) testing substantiallyfree of contamination.

The ability of cells within TRCs to form clonogenic colonies compared toBM-MNCs was determined. Both hematopoietic (CFU-GM) and mesenchymal(CFU-F) colonies were monitored (Table 1). As shown in Table 1, whileCFU-F were increased 280-fold, CFU-GM were slightly decreased byculturing.

TABLE 1 BM MNC Input TRC Output (E−06) (E−06) Fold Exp CFU-GM 1.7 1.1 ±0.2 0.7 ± 0.1 CFU-F 0.03 6.7 ± 1.3 280 ± 67  Results are the average ±SEM from 8 clinical-scale experiments.

The cells of the TRC composition have been characterized by cell surfacemarker expression. Table 2 shows the typical phenotype measured by flowcytometry for starting BM MNCs and TRCs. (See, Table 2) These phenotypicand functional differences highly differentiate TRCs from themononuclear cell starting compositions.

TABLE 2 BM MNC Input TRC Output Total (in Total (in Fold Lineage Marker% millions) % millions) Expansion M CD105/166 0.03 0.1 12 16 373 HCD14auto+ 0.2 0.5 26 36 81 M CD90 0.4 0.9 22 28 39 H (E) CXCR4/ 0.7 1.912 9.9 21 VEGFR1 E CD144/146 0.5 1.3 2.7 3.2 6.3 E VEGFR1 7.6 22 26 382.3 E VEGFR2 12 37 25 37 1.3 H CD14auto− 11 31 14 17 0.9 H CD11b 59 16264 83 0.5 H CD45 97 269 80 104 0.4 H CD3 24 67 8.6 11 0.2 M =mesenchymal lineage, H = hematopoietic lineage, E = endothelial lineage.Results are the average of 4 clinical-scale experiments.

Markers for hematopoietic, mesenchymal, and endothelial lineages wereexamined. Average results from 4 experiments comparing starting BM MNCand TRC product are shown in FIG. 38. Most hematopoietic lineage cells,including CD11b myeloid, CD14auto-monocytes, CD34 progenitor, and CD3lymphoid, are decreased slightly, while CD14auto+ macrophages, areexpanded 81-fold. The mesenchymal cells, defined by CD90+ andCD105+/166+/45−/14− have expansions up to 373-fold. Cells that may beinvolved in vascularization, including mature vascular endothelial cells(CD144/146) and CXCR4/VEGFR1₊ supportive cells are expanded from 6- to21-fold.

Although most hematopoietic lineage cells do not expand in thesecultures, the final product still contains close to 80% CD45+hematopoietic cells and approximately 20% CD90+ mesenchymal cells (FIG.39).

The TRC are highly enriched for CD90⁺ cells compared to the mononuclearcell population from which they are derived. The cells in the TRCcomposition are at least 5%, 10%, 25%, 50%, 75%, or more CD90⁺. Theremaining cells in the TRC composition are CD45⁺. Preferably, the cellsin the TRC composition are about 5-75% viable CD90^(+.) In variousaspects, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more ofthe CD90⁺ are also CD15⁺. (See Table 3) In addition, the CD90⁺ are alsoCD105⁺.

TABLE 3 TRC TRC Run 1 Run 2 % CD90+ 29.89 18.08 % CD90+ CD15− 10.87 3.18% CD90+ CD15+ 19.02 14.90 % CD15+ of the CD90s 63.6 82.4

In contrast, the CD90 population in bone marrow mononuclear cells(BMMNC) is typically less than 1% with the resultant CD45⁺ cells makingup greater than 99% of the nucleated cells in BMMNCs Thus, there is asignificant reduction of many of the mature hematopoietic cells in theTRC composition compared to the starting mononuclear cellpopulation.(See Table 2)

This unique combination of hematopoietic, mesenchymal and endothelialstems cells are not only distinct from mononuclear cells but also othercell populations currently being used in cell therapy. Table 4demonstrates the cell surface marker profile of TRC compared tomesenchymal stem cells and adipose derived stem cells. (Deans R J,Moseley A B. 2000. Exp. Hematol. 28: 875-884; Devine S M. 2002. J CellBiochem Supp 38: 73-79; Katz A J, et al. 2005. Stem Cells. 23:412-423;Gronthos S, et al. 2001. J Cell Physiol 189:54-63; Zuk P A, et al. 2002.Mol Biol Cell. 13: 4279-95.)

For example, Mesenchymal stem cells (MSCs) are highly purified for CD90⁺(greater than 95% CD90⁺), with very low percentage CD45⁺ (if any).Adipose-derived stem cells are more variable but also typically havegreater than 95% CD90⁺, with almost no CD45⁺ blood cells as part of thecomposition. There are also Multi-Potent Adult Progenitor Cells (MAPCs),which are cultured from BMMNCs and result in a pure CD90 populationdifferent from MSCs that co-expresses CD49c. Other stem cells being usedare highly purified cell types including CD34⁺ cells, AC133⁺ cells, and⁺CD34⁺Lin⁻ cells, which by nature have little to no CD90⁺ cells as partof the composition and thus are substantially different from TRCs.

Cell marker analysis have also demonstrated that the TRCs isolatedaccording to the methods of the invention have higher percentages ofCD14⁺ auto⁺, CD34⁺ and VEGFR⁺ cells.

TABLE 4 Adipose- Mesenchymal Derived Stem CD Locus Common Name TRC stemcells Cells CD 34 — + − ± CD13 gp150 + Na + CD15 LewisX, SSEA-1 + − −CD11b Mac-1 + − ± CD14 LPS receptor + − − CD235a glycophorin A + Na NaCD45 Leukocyte common + − − antigen CD90 Thy1 + + + CD105 Endoglin + + +CD166 ALCAM + + + CD44 Hyaluronate receptor + + + CD133 AC133 + − ± —vWF + Na Na CD144 VE-Cadherin + − + CD146 MUC18 +    + • Na CD309VEGFR2, KDR + Na Na

Each of the cell types present in a TRC population have varyingimmunomodulatory properties. Monocytes/macrophages (CD45⁺, CD14⁺)inhibit T cell activation, as well as showing indoleamine2,3-dioxygenase (DO) expression by the macrophages. (Munn D. H. andMellor A. L., Curr Pharm Des., 9:257-264 (2003); Munn D. H., et al. JExp Med., 189:1363-1372 (1999); Mellor A. L. and Munn D. H., J.Immunol., 170:5809-5813 (2003); Munn D H., et al., J. Immunol.,156:523-532 (1996)). The monocytes and macrophages regulate inflammationand tissue repair. (Duffield J. S., Clin Sci (Loud), 104:27-38 (2003);Gordon, S.; Nat. Rev. Immunol., 3:23-35 (2003); Mosser, D. M., J.Leukoc. Biol., 73:209-212 (2003); Philippidis P., et al., Circ. Res.,94:119-126 (2004). These cells also induce tolerance and transplantimmunosuppression. (Fandrich F et al. Hum. Immunol., 63:805-812 (2002)).Regulatory T-cells (CD45⁺ CD4⁺ CD25⁺) regulate innate inflammatoryresponse after injury. (Murphy T.J., et al., J. Immunol., 174:2957-2963(2005)). The T-cells are also responsible for maintenance of selftolerance and prevention and suppression of autoimmune disease.(Sakaguchi S. et al., Immunol. Rev., 182:18-32 (2001); Tang Q., et al.,J. Exp. Med., 199:1455-1465 (2004)) The T-cells also induce and maintaintransplant tolerance (Kingsley C. I., et al. J. Immunol., 168:1080-1086(2002); Graca L., et al., J. Immunol., 168:5558-5565 (2002)) and inhibitgraft versus host disease (Ermann J., et al., Blood, 105:2220-2226(2005); Hoffmann P., et al., Curr. Top. Microbiol. Immunol., 293:265-285(2005); Taylor P. A., et al., Blood, 104:3804-3812 (2004). Mesenchymalstem cells (CD45⁺ CD90⁺ CD105⁺) express IDO and inhibit T-cellactivation (Meisel R., et al., Blood, 103:4619-4621 (2004); Krampera M.,et al., Stem Cells, (2005)) as well as induce anti-inflammatory activity(Aggarwal S. and Pittenger M. F., Blood, 105:1815-1822 (2005)).

TRCs also show increased expression of programmed death ligand 1 (PDL1).Increased expression of PDL1 is associated with production of theanti-inflammatory cytokine IL-10. PDL1 expression is associated with anon-inflammatory state. TRCs have increased PDL1 expression in responseto inflammatory induction, showing another aspect of theanti-inflammatory qualities of TRCs.

TRCs, in contrast to BM MNCs also produce at least five distinctcytokines and one regulatory enzyme with potent activity both for woundrepair and controlled down-regulation of inflammation. (FIG. 40)Specifically, TRCs produce 1) Interleukin-6 (IL-6), 2) Interleukin-10(IL-10), 3) vascular endothelial growth factor (VEGF), 4) monocytechemoattractant protein-1 (MCP-1) and, 5) interleukin-1 receptorantagonist (IL-1ra). The characteristics of these five cytokines issummarized in Table 5, below.

TABLE 5 Characteristics of TRC Expressed Cytokines. CYTOKINECHARACTERISTIC IL-1 ra Decoy receptor for IL-1 down-regulatesinflammation. IL-1 ra and IL-10 are characteristically produced byalternatively activated macrophages IL-6 Interleukin-6 (IL-6) is apleiotropic cytokine with a wide range of biological activities. Thiscytokine regulates polarization of naive CD4⁺ T-cells toward the Th2phenotype, further promotes Th2 differentiation by up-regulating NFAT1expression and inhibits proinflammatory Thl differentiation by inducingsuppressor of cytokine signaling SOCS1. IL-10 Produced by cell typesmediating anti-inflammatory activities, Th2 type immunity,immunosuppression and tissue repair. IL-10 and IL-1ra arecharacteristically produced by alternatively activated macrophages.IL-10 also is involved in the induction of regulatory T-cells. Inaddition, regulatory T-cells secrete high levels of IL-10. MCP-1 MCP-1inhibits the adoptive transfer of autoimmune disease in animal modelsand drives TH2 differentiation indicating an anti inflammatory propertyparticularly when balanced a against MIP-1α. VEGF Angiogenic cytokinewith simultaneous immunosuppressive properties acting at the level ofthe antigen presenting cell.

Additional characteristics of TRCs include a failure spontaneously toproduce, or very low-level production of certain pivotal mediators knownto activate the TH1 inflammatory pathway including interleukin-alpha(IL-1α), interleukin-beta (IL-1β) interferon-gamma (IFNγ) and mostnotably interleukin-12 (IL-12). Importantly, the TRCs neither producethese latter TH1-type cytokines spontaneously during medium replacementor perfusion cultures nor after intentional induction with knowninflammatory stimuli such as bacterial lipopolysaccharide (LPS). TRCsproduced low levels of IFNγ only after T-cell triggering by anti-CD3mAb. Finally, the TRCs produced by the current methods produce more ofthe anti-inflammatory cytokines IL-6 and IL-10 as well as less of theinflammatory cytokine IL-12.

Moreover, TRCs are inducible for expression of a key immune regulatoryenzyme designated indoleamine-2,-3 dioxygenase (IDO). The TRCs accordingto the present invention express higher levels of IDO upon inductionwith interferon y. IDO has been demonstrated to down-regulate bothnascent and ongoing inflammatory responses in animal models and humans(Meisel R., et al., Blood, 103:4619-4621 (2004); Munn D. H., et al., J.Immunol., 156:523-532 (1996); Munn D. H., et al. J. Exp. Med.189:1363-1372 (1999); Munn D. H. and Mellor A. L., Curr. Pharm. Des.,9:257-264 (2003); Mellor A. L. and Munn D. H., J. Immunol.,170:5809-5813 (2003)).

Together, these unique characteristics of the TRCs according to theinvention create a more anti-inflammatory environment for tissue repair,and therefore are more effective treatment for tissue repair.

As discussed above, TRCs are highly enriched for a population of cellsthat co-express CD90 and CD 15.

CD90 is present on a stem and progenitor cells that can differentiateinto multiple lineages. These cells are a heterogeneous population ofcells that are most likely at different states of differentiation. Cellmarkers have been identified on stem cells of embryonic or fetal originthat define the stem-cell state of the cell. One of these markers,SSEA-1, also referred to as CD15. CD15 is found on mouse embryonic stemcells, but is not expressed on human embryonic stem cells. It hashowever been detected in neural stem cells from both mouse and human.CD15 is also not expressed on purified mesenchymal stem cells derivedfrom human bone marrow or adipose tissue (see Table 6). Thus, the cellpopulation in TRCs that co-express both CD90 and CD15 are a unique cellpopulation and may define a the stem-like state of the CD90adult-derived cells.

Accordingly, in another aspect of the invention the cell populationexpressing both CD90 and CD15 may be further enriched. By furtherenriched is meant that the cell composition contains 5%, 10%, 25%, 50%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100% CD90⁺ CD15⁺ cells.TRCs can be further enriched for CD90⁺ CD15⁺ cells by methods known inthe art such as positive or negative selection using antibodies directto cell surface markers. The TRCs that have been further enriched forCD90⁺ CD15⁺ cells are particularly useful in bone repair andregeneration.

TABLE 6 Cell Phenotype TRC MSC P0 % CD90+ 23.99 98.64 % CD15+ 39.89 0.76% CD15+/CD90+ 19.54 0.22 N 2 4

The CFU-F and osteogenic potential of CD90⁺ CD15⁺ was assessed. WhenCD90⁺ cells are removed, all CFU-F and in vitro osteogenic potential isdepleted. Suprisingly, although the overall frequency of CD90 and CFU-Fare higher in MSC cultures (where CD90 do not express CD15), therelative number of CFU-F per CD90 cells is dramtically higher in TRC.(FIG. 41) This demonstrates that the CD90 cells are much more potent inTRCs that when grown as purified cell polulations.

Osteogenic potential was measured both in vitro and in vivo. Again, inconditions where cells are expressing CD15 (TRC), the osteogenicpotential was higher than that found in mesenchymal cells (FIG. 42)

Therapeutic Methods

Tissue Repair Cells (TRCs) are useful for a variety of therapeuticmethods including, tissue repair, tissue regeneration, and tissueengineering. For example, the TRC are useful in bone regeneration,cardiac regeneration, vascular regeneration, neural regeneration and thetreatment of ischemic disorders. Ischemic conditions include, but arenot limited to, limb ischemia, congestive heart failure, cardiacischemia, kidney ischemia and ESRD, stroke, and ischemia of the eye.Additionally, because of the immuno-regulatory cytokines produced by theTRCs, the TRCs are also useful in the treatment of a variety of immuneand inflammatory disease. Immune and inflammatory disease include forexample, diabetes (Type I and Type II), inflammatory bowel diseases(IBD), graft verses host disease (GVHD), psoriasis, rejection ofallogeneic cells, tissues or organs (tolerance induction), heartdisease, spinal cord injury, rheumatoid arthritis, osteo-arthritis,inflammation due to hip replacement or revision, Crohn's disease,autoimmune diseases such as system lupus erythematosus (SLE), rheumatoidarthritis (RA), and multiple sclerosis (MS). In another aspect of theinvention TRCs are also useful for inducing angiogenesis.

TRCs are administered to mammalian subjects, e.g., human, to effecttissue repair or regeneration. The TRCs are administered allogeneicallyor autogeneically.

TRCs unique qualities strongly polarize the host response away from thetissue-destructive pathway of inflammation and toward wound repair withrapid healing of injured tissues. Furthermore, some of the cells arecapable of tissue specific differentiation (e.g., CD90⁺ to bone),further aiding tissue regeneration. Accordingly, TRCs are effective forinducing tissue repair in a wide range of diseases.

Pharmaceutical Administration and Dosage Forms

The described TRCs can be administered as a pharmaceutically orphysiologically acceptable preparation or composition containing aphysiologically acceptable carrier, excipient, or diluent, andadministered to the tissues of the recipient organism of interest,including humans and non-human animals. TRC-containing composition canbe prepared by resuspending the cells in a suitable liquid or solutionsuch as sterile physiological saline or other physiologically acceptableinjectable aqueous liquids. The amounts of the components to be used insuch compositions can be routinely determined by those having skill inthe art.

The TRCs or compositions thereof can be administered by placement of theTRC suspensions onto absorbent or adherent material, i.e., a collagensponge matrix, and insertion of the TRC-containing material into or ontothe site of interest. Alternatively, the TRCs can be administered byparenteral routes of injection, including subcutaneous, intravenous,intramuscular, and intrasternal. Other modes of administration include,but are not limited to, intranasal, intrathecal, intracutaneous,percutaneous, enteral, and sublingual. In one embodiment of the presentinvention, administration of the TRCs can be mediated by endoscopicsurgery.

For injectable administration, the composition is in sterile solution orsuspension or can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures. The amounts or quantities,as well as the routes of administration used, are determined on anindividual basis, and correspond to the amounts used in similar types ofapplications or indications known to those of skill in the art.

Consistent with the present invention, the TRC can be administered tobody tissues, including liver, pancreas, lung, salivary gland, bloodvessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney,muscle, cardiac muscle, nerve, skeletal muscle, joints, and limb.

The number of cells in a TRC suspension and the mode of administrationmay vary depending on the site and condition being treated. Asnon-limiting examples, in accordance with the present invention, about35-300×10⁶ TRCs are injected to effect tissue repair. Consistent withthe Examples disclosed herein, a skilled practitioner can modulate theamounts and methods of TRC-based treatments according to requirements,limitations, and/or optimizations determined for each case.

In preferred embodiments, the TRC pharmaceutical composition comprisesbetween about 8 and 54% CD90⁺ cells and between about 46 and 92% CD45⁺cells. The TRC pharmaceutical composition preferably contains betweenabout 35×10⁶ and 300×10⁶ viable nucleated cells and between about 7×10⁶and 75×10⁶ viable CD90⁺ cells. The TRC pharmaceutical compositionalpreferably has less than 0.5 EU/ml of endotoxin and no bacterial orfungal growth. In preferred embodiments, a dosage form of TRCs iscomprised within 4.7-7.3 mL of pharmaceutically acceptable aqueouscarrier. The preferred suspension solution is Multiple ElectrolyteInjection Type 1 (USP/EP). Each 100 mL of Multiple Electrolyte InjectionType 1 contains 234 mg of Sodium Chloride, USP (NaCl); 128 mg ofPotassium Acetate, USP (C₂H₃KO₂); and 32 mg of Magnesium AcetateTetrahydrate (Mg(C₂H₃O₂)₂.4H₂O). It contains no antimicrobial agents.The pH is adjusted with hydrochloric acid. The pH is 5.5 (4.0 to 8.0).The Multiple Electrolyte Injection Type 1 is preferably supplementedwith 0.5% human serum albumin (USP/EP). Preferably, the TRCpharmaceutical composition is stored at 0-12 ° C., unfrozen.

Indications and Modes of Delivery for TRCs

TRCs may be manufactured and processed for delivery to patients usingthe described processes where the final formulation is the TRCs with allculture components substantially removed to the levels deemed safe bythe FDA. It is critical for the cells to have a final viability greaterthan 70%, however the higher the viability of the final cell suspensionthe more potent and efficacious the final cell dose will be, and theless cellular debris (cell membrane, organelles and free nucleic acidfrom dead cells), so processes that enhance cell viability whilemaintaining the substantially low culture and harvest components, whilemaintaining closed aseptic processing systems are highly desirable.

Limb Ischemia

It has been demonstrated that bone marrow-derived cells are used forvascular regeneration in patients with critical limb ischemia,peripheral vascular disease, or Burger's syndrome. The TRCs delivered topatients with ischemic limbs, and have been shown to enhance vascularregeneration. TRCs are delivered to patients by creating a cellsuspension and removing the TRCs from the supplied bag or vial that theyare delivered in. A syringe is used to remove the TRC suspension, andthen smaller 0.25 ml to 1 ml individual injection volumes are loadedfrom the main syringe using a syringe adaptor, and then severalindividual injection volumes are delivered via intramuscular injectionto the site of limb ischemia and where vascular formation is required.The TRCs may be delivered through a wide range of needle sizes, fromlarge 16 gauge needles to very small 30 gauge needles, as well as verylong 28 gauge catheters for minimally invasive procedures.Alternatively, the TRCs may also be delivered intravascularly andallowed to home to the site of ischemia to drive local tissueregeneration.

Cardiac Regeneration

There are a variety of modes of delivery for driving cardiac tissueregeneration. The TRCs are delivered intra-vascularly and allowed tohome to the site of regeneration. Alternatively, the TRCs are also bedelivered directly into the cardiac muscle, either epicardially orendocardially, as well as transvascularly. The TRCs may be deliveredduring an open-chest procedure, or via minimally invasive proceduressuch as with delivery via catheter. The TRCs are delivered to thesepatients by creating a cell suspension and removing the TRCs from thesupplied bag or vial that they are delivered in. A syringe is used toremove the TRC suspension, and then smaller 0.25 ml to 1 ml individualinjection volumes are loaded from the main syringe using a syringeadaptor, and then several individual injection volume are delivered viaintramuscular injection to the site of cardiac ischemia and wherevascular formation is required. The TRCs may be delivered through a widerange of needle sizes, from large 16 gauge needles to very small 30gauge needles, as well as very long 28 gauge catheters for minimallyinvasive procedures.

Spinal Cord Regeneration

There are a variety of ways that TRCs are used for regeneration afterspinal cord injury (SCI): TRCs may be injected directly into the site ofSCI, seeded onto a matrix (chosen from the list below for boneregeneration) and seeded into resected spinal cord or just placed at thesite such that the TRCs may migrate to the injury site. Alternatively,the TRCs are delivered intravascularly and allowed to home to the siteof injury to drive local tissue regeneration.

There are a variety of other applications where the TRCs may bedelivered locally to the tissue via direct injection, seeding onto amatrix for localized delivery, or delivered via the vascular systemallowing for TRCs to home to the site of injury or disease. Thesediseases are limb ischemia, congestive heart failure, cardiac ischemia,kidney ischemia and end stage renal disease, stroke, and ischemia of theeye.

Orthopedic Indications for Bone Regenerations

TRCs have been used successfully in bone regeneration applications inhumans. Optionally, TRCs are mixed with 3D matrices to enhance deliveryand localization at the site where bone regeneration is required. Thethree-dimensional matrices come in a range of physical and chemicalforms, and viscous or gelled binding materials may also be added to aidhandling and delivery properties.

Three dimensional matrices include for example, demineralized boneparticles, mineralized bone particles, synthetic ceramic of the calciumphosphate family such as alpha tri-calcium phosphates (TCP), beta TCP,hydroxyappatites, and complex mixtures of these materials. Othermatrices include for example, collagen-based sponges,polysaccharide-based materials such as hyaluronan and alginates,synthetic biodegradable polymeric materials such as poly-lactides,poly-glycolides, poly-fumarates, poly-ethylene glycol, co-polymers ofthese as well as other materials known in the art.

Any of the matrices used with TRCs may be processed into differentphysical forms that are common in the art for tissue regenerationapplications. These physical forms are open and closed pore foams andsponges, fiber-based woven or non-woven meshes, or small particlesranging from nano-particles to micron-sized particles (1 micrometer-1000micrometers) and macro-particles in the millimeter size scale. The smallparticles also often have an open porosity, with nanopores aiding innutrient and metabolite transport and micropores providing pores largeenough to facilitate cell seeding and tissue integration.

When the matrices used for cell delivery are small particles deliveredto wound sites, at times viscous materials or gels are used to bind theparticles that aid in materials handling and delivery, as well ashelping to keep the particles and the cells localized at the site afterplacement. Viscous binding materials include for example, hyaluronan,alginates, collagens, poly ethylene glycols, poly fumarates, blood clotsand fibrin-based clots, as well as mixtures of these materials, eitherin the form of viscous fluids to soft or hard hydrogels. Other viscousmaterials and hydrogels are known in the art

In various embodiments, TRCs are delivered with TCP, demineralized bone,and mineralized bone particles in sizes ranging from 200 micrometers to5 millimeters, depending on the specific application. Optionally, thesematerials are bound with fibrin-based clots made from autologous freshlyprepared plasma from the patient. Other fibrin clots or differenthydrogels, or matrix materials common may also be used.

Generally, TRCs are mixed with the matrices just prior to surgery whenused for bone regeneration. For long-bone regeneration, typically thearea of bone non-union is opened by the surgeon, and the necrotic boneis removed. The non-unioned bone or area where bone is needed may or maynot be de-corticated by the surgeon to allow bleeding at the site, atwhich point the TRC-matrix mixture is placed by the surgeon between thebones where regeneration will occur. This mixture of the TRCs and matrixdrive tissue regeneration with the physical matrix guiding the locationof bone regeneration and the TRCs providing the tissue repair stimulusfor driving angiogenesis, would healing, and bone regeneration. Theremaining TRC/matrix mixture is optionally placed around the fractureline after any orthopedic hardware has been placed such as plates, rods,screws or nails.

Methods of Production of TRCs

TRCs are isolated from any mammalian tissue that contains bone marrowmononuclear cells (BM MNC). Suitable sources for BM MNC is peripheralblood, bone marrow, umbilical cord blood or fetal liver. Blood is oftenused because this tissue is easily obtained. Mammals include forexample, a human, a primate, a mouse, a rat, a dog, a cat, a cow, ahorse or a pig.

The culture method for generating TRCs begins with the enrichment of BMMNC from the starting material (e.g., tissue) by removing red bloodcells and some of the polynucleated cells using a conventional cellfractionation method. For example, cells are fractionated by using aFICOLL® density gradient separation. The volume of starting materialneeded for culture is typically small, for example, 40 to 50 mL, toprovide a sufficient quantity of cells to initiate culture. However, anyvolume of starting material may be used.

Nucleated cell concentration is then assessed using an automated cellcounter, and the enriched fraction of the starting material isinoculated into a biochamber (cell culture container). The number ofcells inoculated into the biochamber depends on its volume. TRC cultureswhich may be used in accordance with the invention are performed at celldensities of from 10⁴ to 10⁹ cells per ml of culture. When a AastromReplicell Biochamber is used 2-3×10⁸ total cells are inoculated into avolume of approximately 280 mL.

Prior to inoculation, a biochamber is primed with culture medium.Illustratively, the medium used in accordance with the inventioncomprises three basic components. The first component is a mediacomponent comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha Medium orMcCoy's Medium, or an equivalent known culture medium component. Thesecond is a serum component which comprises at least horse serum orhuman serum and may optionally further comprise fetal calf serum,newborn calf serum, and/or calf serum. Optionally, serum free culturemediums known in the art may be used. The third component is acorticosteroid, such as hydrocortisone, cortisone, dexamethasone,solumedrol, or a combination of these, preferably hydrocortisone. Whenthe Aastrom Replicell Biochamber is used, the culture medium consists ofIMDM, about 10% fetal bovine serum, about 10% horse serum, about 5 μMhydrocortisone, and 4 mM L-Glutamine. The cells and media are thenpassed through the biochamber at a controlled ramped perfusion scheduleduring culture process. The cells are cultures for 2, 4, 6, 8, 10, 12,14, 16 or more days. Preferably, the cells are cultured for 12 days. Forexample, when used with the Aastrom Replicell System Cell Cassette, thecultures are maintained at 37 ° C. with 5% CO₂ and 20% O₂.

These cultures are typically carried out at a pH which is roughlyphysiologic, i.e. 6.9 to 7.6. The medium is kept at an oxygenconcentration that corresponds to an oxygen-containing atmosphere whichcontains from 1 to 20 vol. percent oxygen, preferably 3 to 12 vol.percent oxygen. The preferred range of O₂ concentration refers to theconcentration of O₂ near the cells, not necessarily at the point of O₂introduction which may be at the medium surface or through a membrane.

Standard culture schedules call for medium and serum to be exchangedweekly, either as a single exchange performed weekly or a one-halfmedium and serum exchange performed twice weekly. Preferably, thenutrient medium of the culture is replaced, preferably perfused, eithercontinuously or periodically, at a rate of about 1 ml per ml of cultureper about 24 to about 48 hour period, for cells cultured at a density offrom 2×10⁶ to 1×10⁷ cells per ml. For cell densities of from 1×10⁴ to2×10⁶ cells per ml the same medium exchange rate may be used. Thus, forcell densities of about 10⁷ cells per ml, the present medium replacementrate may be expressed as 1 ml of medium per 10⁷ cells per about 24 toabout 48 hour period. For cell densities higher than 10⁷ cells per ml,the medium exchange rate may be increased proportionality to achieve aconstant medium and serum flux per cell per unit time

A method for culturing bone marrow cells is described in Lundell, etal., “Clinical Scale Expansion of Cryopreserved Small Volume Whole BoneMarrow Aspirates Produces Sufficient Cells for Clinical Use,” J.Hematotherapy (1999) 8:115-127 (which is incorporated herein byreference). Bone marrow (BM) aspirates are diluted in isotonic bufferedsaline (Diluent 2, Stephens Scientific, Riverdale, N.J.), and nucleatedcells are counted using a Coulter ZM cell counter (Coulter Electronics,Hialeah, Fla.). Erythrocytes (non-nucleated) are lysed using a ManualLyse (Stephens Scientific), and mononuclear cells (MNC) are separated bydensity gradient centrifugation (Ficoll-Paque® Plus, Pharmacia Biotech,Uppsala, Sweden) (specific gravity 1.077) at 300 g for 20 min at 25° C.BM MNC are washed twice with long-term BM culture medium (LTBMC) whichis Iscove's modified Dulbecco's medium (IMDM) supplemented with 4 mML-glutamine 9GIBCO BRL, Grand Island, N.Y.), 10% fetal bovine serum(FBS), (Bio-Whittaker, Walkersville, Md.), 10% horse serum (GIBCO BRL),20 μg/ml vancomycin (Vancocin® HCl, Lilly, Indianapolis, Ind.), 5 μg/mlgentamicin (Fujisawa USA, Inc., Deerfield, Ill.), and 5 μMhydrocortisone (Solu-Cortef®, Upjohn, Kalamazoo, Mich.) before culture.

Cell Storage

After culturing, the cells are harvested, for example using trypsin, andwashed to remove the growth medium. The cells are resuspended in apharmaceutical grade electrolyte solution, for example Isolyte (B. BraunMedical Inc., Bethlehem, Pa.) supplemented with serum albumin.Alternatively, the cells are washed in the biochamber prior to harvestusing the wash harvest procedure described below. Optionally afterharvest the cells are concentrated and cryopreserved in a biocompatiblecontainer, such as 250 ml cryocyte freezing containers (BaxterHealthcare Corporation, Irvine, Calif.) using a cryoprotectant stocksolution containing 10% DMSO (Cryoserv, Research Industries, Salt LakeCity, Utah), 10% HSA (Michigan Department of gPublic Health, Lansing,MI), and 200 μg/ml recombinant human DNAse (Pulmozyme®, Genentech, Inc.,South San Francisco, Calif.) to inhibit cell clumping during thawing.The cryocyte freezing container is transferred to a precooled cassetteand cryopreserved with rate-controlled freezing (Model 1010, FormaScientific, Marietta, Ohio). Frozen cells are immediately transferred toa liquid nitrogen freezer (CMS-86, Forma Scientific) and stored in theliquid phase. Preferred volumes for the concentrated cultures range fromabout 5 mL to about 15 ml. More preferably, the cells are concentratedto a volume of 7.5 mL.

Post-Culture

When harvested from the biochamber the cells reside in a solution thatconsists of various dissolved components that were required to supportthe culture of the cells as well as dissolved components that wereproduced by the cells during the culture. Many of these components areunsafe or otherwise unsuitable for patient administration. To createcells ready for therapeutic use in humans it is therefore required toseparate the dissolved components from the cells by replacing theculture solution with a new solution that has a desired composition,such as a pharmaceutical-grade, injectable, electrolyte solutionsuitable for storage and human administration of the cells in a celltherapy application.

A significant problem associated with many separation processes iscellular damage caused by mechanical forces applied during theseprocesses, exhibited, for instance, by a reduction in viability andbiological function of the cells and an increase in free cellular DNAand debris. Additionally, significant loss of cells can occur due to theinability to both transfer all the cells into the separation apparatusas well as extract all the cells from the apparatus.

Separation strategies are commonly based on the use of eithercentrifugation or filtration. An example of centrifugal separation isthe COBE 2991 Cell Processor (COBE BCT) and an example of a filtrationseparation is the CYTOMATE® Cell Washer (Baxter Corp) (Table 7). Bothare commercially available state-of-the-art automated separation devicesthat can be used to separate (wash) dissolved culture components fromharvested cells. As can be seen in Table 7, these devices result in asignificant drop in cell viability, a reduction in the total quantity ofcells, and a shift in cell profile due to the preferential loss of thelarge and fragile CD14⁺auto⁺ subpopulation of TRCs.

TABLE 7 Performance of 2 different cell separation devices, 3 differentstudies. COBE 2991 Cell CYTOMATE ® Cell CYTOMATE ® Cell Processor (n =3) Washer (n = 8) Washer (n = 26) Operating principal CentrifugationFiltration Filtration Study Reference Aastrom internal protocol Aastromnew wash US Fracture Clinical report #PABI0043 process development,Trial, BB-IND #10486 report MF#0384 Average pre-separation 93% 93% 95%cell viability Average post-separation 83% 71% 81% cell viabilityAverage reduction in 18% 69% Not available CD14⁺ Auto⁺ frequency Averagecell recovery 73% 74% Not available

These limitations in the art create difficulties in implementingmanufacturing and production processes for creating cell populationssuitable for human use. It is desirable for the separation process tominimize damage to the cells and thereby result in a cell solution thatis depleted of unwanted dissolved components while retaining highviability and biological function with minimal loss of cells.Additionally, it is important to minimize the risk of introducingmicrobial contaminants that will result in an unsafe final product. Lessmanipulation and transfer of the cells will inherently reduce this risk.

The invention described in this disclosure overcomes all of theselimitations in the current art by implementing a separation process towash the cells that minimizes exposure of the cells to mechanical forcesand minimizes entrapment of cells that cannot be recovered. As a result,damage to cells (e.g. reduced viability or function), loss of cells, andshift in cell profile are all minimized while still effectivelyseparating unwanted dissolved culture components. In a preferredimplementation, the separation is performed within the same device thatthe cells are cultured in which eliminates the added risk ofcontamination by transfer and separation using another apparatus. Thewash process according to the invention is described below.

Wash Harvest

As opposed to conventional culture processes where cells are removed(harvested) from the biochamber followed by transfer to anotherapparatus to separate (wash) the cells from culture materials, thewash-harvest technique reverses the order and provides a unique means tocomplete all separation (wash) steps prior to harvest of the cells fromthe biochamber.

To separate the culture materials from the cells, a new liquid ofdesired composition (or gas) may be introduced, preferably at the centerof the biochamber and preferably at a predetermined, controlled flowrate. This results in the liquid being displaced and expelled along theperimeter of the biochamber, for example, through apertures 48, whichmay be collected in the waste bag 76.

In some embodiments of the invention, the diameter of the liquid spacein the biochamber is about 33 cm, the height of the liquid space isabout 0.33 cm and the flow rates of adding rinsing and/or harvestingfluids to the biochamber is about 0.03 to 1.0 volume exchanges (VE) perminute and preferably 0.50 to about 0.75 VE per minute. Thissubstantially corresponds to about 8.4 to about 280 mL/min andpreferably 140 to about 210 ml/min. The flow rates and velocities,according to some embodiments, aid in insuring that a majority of thecultured cells are retained in the biochamber and not lost into thewaste bag and that an excessively long time period is not required tocomplete the process. Generally, the quantity of cells in the chambermay range from 10⁴ to 10⁸ cell/mL. For TRCs, the quantity may range from10⁵ to 10⁶ cells/mL, corresponding to 30 to 300 million total cells forthe biochamber dimensions above. Of course, one of skill in the art willunderstand that cell quantity changes upon a change in the biochamberdimensions

According to some embodiments, in harvesting the cultured cells from thebiochamber, the following process may be followed, and is broadlyoutlined in Table 3, below. The solutions introduced into the biochamberare added into the center of the biochamber. The waste media bag 76 maycollect corresponding fluid displaced after each step where a fluid orgas is introduced into the biochamber. Accordingly, after cells arecultured, the biochamber is filled with conditioned culture medium(e.g., IMDM, 10% FBS, 10% Horse Serum, metabolytes secreted by the cellsduring culture) and includes between about 30 to about 300 millioncells. A 0.9% NaCl solution (“rinse solution”) may then be introducedinto the biochamber at about 140 to 210 mL per minute until about 1.5 toabout 2.0 liters of total volume has been expelled from the biochamber(Step 1).

While a single volume exchange for introduction of a new or differentliquid within the biochamber significantly reduces the previous liquidwithin the biochamber, some amount of the previous liquid will remain.Accordingly, additional volume exchanges of the new/different liquidwill significantly deplete the previous liquid.

Optionally, when the cells of interest are adherent cells, such as TRCs,the rinse solution is replaced by harvest solution. A harvest solutionis typically an enzyme solution that allows for the detachment of cellsadhered to the culture surface. Harvest solutions include for example0.4% Trypsin/EDTA in 0.9% NaCl that may be introduced into thebiochamber at about 140 to 210 mL per minute until about 400 to about550 ml of total volume has been delivered (Step 2). Thereafter, apredetermined period of time elapses (e.g., 13-17 minutes) to allowenzymatic detachment of cells adhered to the culture surface of thebiochamber (Step 3).

Isolyte (B Braun) supplemented with 0.5% HSA may be introduced at about140 to 210 mL per minute until about 2 to about 3 liters of total volumehas been delivered, to displace the enzyme solution (Step 4).

At this point, separation of unwanted solutions (culture medium, enzymesolution) from the cells is substantially complete.

To reduce the volume collected, some of the Isolyte solution ispreferably displaced using a gas (e.g., air) which is introduced intothe biochamber at a disclosed flow rate (Step 5). This may be used todisplace approximately 200 to 250 cc of the present volume of thebiochamber.

The biochamber may then be agitated to bring the settled cells intosolution (Step 6). This cell suspension may then be drained into thecell harvest bag 70 (or other container) (Step 7). An additional amountof the second solution may be added to the biochamber and a secondagitation may occur in order to rinse out any other residual cells(Steps 8 & 9). This final rinse may then be added to the harvest bag 70(Step 10).

TABLE 8 Wash-harvest Protocol Step Number & Name Description 1 Rinse outculture media Use Sodium Chloride to displace the culture medium intothe waste container. 2 Add Trypsin solution Replace Sodium Chloride inculture chamber with the Trypsin solution. 3 Trypsin incubation Static15 minute incubation in Trypsin solution. 4 Rinse out Trypsinsolution/Transfer Add Isolyte with 0.5% HSA to displace the Trypsin inPharmaceutically Acceptable solution into the waste container. Carrier 5Concentration/Volume reduction Displace some of the Isolyte solutionwith air to reduce the final volume (concentration step) 6 AgitateBiochamber Rocking motion to dislodge and suspend cells into Isolytesolution for collection 7 Drain into Collection Container Drain Cells inIsolyte solution into cell collection bag. 8 Add rinse solution toBiochamber Add more Isolyte to rinse out residual cells. 9 AgitateBiochamber Rocking motion to dislodge and suspend cells into Isolytesolution for collection 10 Drain into Collection Container Drain thefinal rinse into the cell collection bag.

Compared to TRCs produced using a conventional method for post-culturewash (e.g. CYTOMATE®), TRCs produced using the wash-harvest process showhigher and more consistent post-wash viability, higher post-storageviability, higher total viable cell number, higher total viable CD90⁺cell number, slightly lower residual BSA, and higher and more consistentCFU-F and CFU-GM per product. The post wash viability is more consistentwith the new wash process with a standard deviation of 2% as compared to10% for the CYTOMATE® wash process. TRCs produced using the wash-harvestalso have a higher percentage of CD90⁺ cells, meaning that there is ahigher percentage of marrow stromal cells in the TRCs as well as CD14⁺cells, meaning there is a higher percentage of monocyte/macrophage cellsin the TRCs. The presence of VEGFR1 was also increased in wash-harvestTRCs. Although the final viable total cell number is higher with the newwash process, the new wash product contains more non-dissociatedaggregates of viable cells which should be distinguished from aggregatesdue to debris—a likely source of the large cell clumps occasionally seenin the CYTOMATE® wash product after 24 hours of storage. Thesenon-dissociated aggregates do not appear to interfere with cell productstorage or delivery.

Methods of Separation

The wash-harvest process described above is also useful for theseparation of solutions with dissolved components from particlescontained within the solution. The wash-harvest process according to theinvention is based on the unexpected ability to generate a controlledflow of solution over particles settled on a horizontal surface suchthat the particles are not removed by the flow and effluent solution iscollected free of particles.

The process uses, for example, a thin cylindrical chamber with itsdiameter oriented horizontally and with a height that is sufficientlysmall so that a solution introduced to an empty chamber will fill theheight before flowing horizontally. The diameter of the chamber issufficiently large to accommodate the quantity of particles to beseparated and is typically many times the height of the chamber.

Typically, the chamber includes a height of about 0.4 cm to match thedesired height for use of the chamber for culture of cells, but may bein the range of about 0.2 to about 1.0 cm (or more). The diameter of thechamber may be about 33 cm, but may also include a range of about 10 cmto about 50 cm (or more). Accordingly, a preferable chamber volume,according to some embodiments, may be about 280 cc, though such volume(of course) corresponds to the ranges of chamber diameter and heights.

Prior to the start of the separation process, the chamber volume iscompletely filled with a first solution containing particles. Theparticles are of higher density than the solution and are settled bygravity or adhered on the bottom circular surface of the chamber. Thetotal stacked height of the particles in the chamber, which can beminimized by uniform distribution of the particles across the bottomsurface, is a small fraction of the total height of the chamber. Toperform the separation, a second solution of a desired new compositionis introduced at the center of the chamber at a controlled flow rate andthe solution flows symmetrically toward the perimeter of the chamber,displacing the first solution in the chamber which flows out of thechamber at the perimeter and is directed to a common collection point.As a result of the geometry, the linear velocity of the flow decreasesin proportion to the distance from the center such that the linearvelocity is slowest where it exits the perimeter of the chamber. Theflow rate is preferably controlled so that the linear velocity issufficiently low to prevent movement of settled particles out of thechamber, but only remove liquid therein. The relatively small height ofthe biochamber as described herein preferably allows for a plug-flow inthe radial direction to minimize mixing of the displaced solution withthe new solution. Accordingly, this allows a high percentage of thefirst solution to be displaced with a second solution from thebiochamber. One or more additional volume exchanges with the secondsolution can be performed to further reduce residual levels of the firstsolution within the chamber.

As an alternative operating mode prior to removing the particles fromthe chamber, the first solution can also be displaced by a gas, such asair, that is introduced at the center of the chamber and within the sameflow rate range as described for introduction of a second solution. Thisresults in a controlled reduction of the volume of liquid within thechamber while still retaining the particles. A rinse solution of asmaller volume than the chamber can then be introduced as a carrier toremove and collect the particles from the chamber in a reduced solutionvolume.

A variety of solutions that are compatible with the contained particlescan be used as the exchange liquid—e.g. for cells: any culture medium,any physiological buffer, any pharmaceutical grade injectable.

Any enzyme solution conventionally used in cell culture for detachmentand harvest of adhered cells can be used. According to the method of theinvention, culture media with contained adhered cells is replaced with abuffer containing the enzyme. The cells are exposed to the enzyme for aperiod of time so that the adherent cells are no longer adherent. Theenzyme buffer is then replaced with another fluid that the cells willeither be stored in or used in. The chamber is then agitated to causethe cells to be suspended in the fluid and the fluid is collected in abiocompatible container. For example, the fluid may be a cryo-protectantfor storage at −80 deg. C. or a pharmaceutically acceptable carrier forpatient administration, Enzyme solutions for cell harvest includetrypsins (animal-derived, microbial-derived, or recombinant), variouscollagenases, alternative microbial-derived enzymes, dissociationagents, general proteases, or mixtures of these. A list of somecommercial harvest enzyme solutions are listed below:

Reagent Manufacturer Description Aastrom Replicell ® Invitrogen Porcinederived Harvest Reagent trypsin TrypLe ™ Invitrogen Recombinant enzymederived from microbial fermentation TrypZean ™ Solution SigmaRecombinant bovine trypsin expressed in corn HyQTase ™ HyCloneProteolytic and collagenolytic enzymes Accutase Innovative CellProteolytic and Technologies, Inc. collagenolytic enzymes AccumaxSolution Innovative Cell Proteolytic and Technologies, Inc.collagenolytic enzymes plus cell dispersal agents Recombinant CascadeBiologics Recombinant bovine Trypsin/EDTA trypsin

Bioreactor System

Some embodiments of the invention include methods and/or devices forcreating post-culture cell compositions suitable for therapeutic use,and may be related to methods and devices/systems disclosed in U.S. Pat.Nos. 6,326,198 and 6,048,721.

For example, the bioreactor system as disclosed in U.S. Pat. No.6,048,721 (the '721 patent) may be used to perform the methods accordingto some embodiments of the invention. A portion of this disclosure,describing a system for carrying embodiments of the present invention isset out below.

As shown in FIG. 1, a bioreactor system includes a disposable cellcassette 100 where the growth and expansion of cells takes places, ahardware incubator unit 200 and companion hardware, a system manager 300that controls the biological and physical environment during theexpansion process, and a processor unit 400 that facilitates at leastone of the filling, processing and inoculation of cells, as well as thefinal harvest of cells at the completion of the expansion process.

Simulating bone marrow for the purpose of ex vivo growth and expansionof mammalian stem and hematopoietic progenitor cells generally requires,amongst other things, a uniform oxygen concentration and a uniformsupply of a nutrient carrying perfusing liquid for all of the cellsbeing cultured. A primary function of the cell cassette 100 is toprovide a steriley closed environment that supports oxygenation andmedium perfusion of the contained biochamber

Referring to FIG. 5, the primary element of the cell cassette is adisc-like bioreactor culture chamber 10 (“biochamber”) having apreferably circular outer periphery. The biochamber may be formed offour main components: a top 20, a base 30, a cell bed disc 40 and a gaspermeable, liquid impervious membrane 50.

As shown schematically in FIG. 7, the top 20 of the biochamber issecured to the base 30 (preferably in a fluid tight manner), for exampleby applying localized energy to weld the two pieces together (or otherfastening means such as a plurality of screws), at its radially outerperiphery. The membrane 50 is clamped between the top and the base andis tightly stretched so as to separate the interior volume of thebiochamber into upper and lower portions.

The cell bed disc 40 is located within the lower portion of the interiorof the biochamber. It has generally a disc shape with an upwardlyextending annular lip 42 at its outer radial periphery. Followinginoculation of starting cells, cell growth occurs in a cell growth bed25 defined between the upper surface of the disc 40, the lower surfaceof the membrane 50 and the annular lip 42. The upper surface A of theannular lip 42 is preferably coplanar with the upper surface of theflange 30 A of the base 30 when the disc 40 is fitted in the base, sothat membrane 50 can cooperate with the lip 42 to seal the cell growthbed 25.

The disc 40 of the cell bed has a radially central growth media supplyport 44, which extends downwardly through the base 30 to the exterior ofthe biochamber. Alternatively, the growth media supply port may belocated at a radially central point above disc 40 of the cell bed andextend upwardly through the raised centerport 28 to the exterior of thebiochamber (not shown). It also has at least one harvest port 46 (mayalso include a plurality of harvest ports) near the radially outer limitof the cell bed, i.e., just inside the lip 42. The port 46 also extendsthrough the base to the exterior of the biochamber. Alternatively, theone or more harvest ports may be located near the radially outer limitof the base, i.e. just inside the perimeter of the cell bed and extenddirectly from the base to the exterior of the biochamber (not shown).Finally, a plurality of, e.g., 24, waste media discharge apertures 48are located at the perimeter of the disc 40 to allow fluid communicatonbetween the cell bed compartment established above the disc and thewaste compartment established below the disc. The apertures 48 arepreferably equally spaced about the radially outer periphery of the disc40, immediately adjacent to the lip 42.

A nutrient rich growth media is supplied to the media supply port 44.The growth media may be a standard growth medium, as is well known inthe art, and may have a serum supplement such as fetal bovine serum,horse serum or human serum. It may also be serum free. Growth factorsand reagents such as glutamine may also be added as necessary. Thegrowth media may be supplied in premixed bags or may be modified onsite.

From the media supply port, the growth media enters the cell bed 25 andflows radially outwardly toward the radial periphery of the disc 40. Asit does so, it supplies nutrients to, and removes waste products from,the cells being cultured therein. It is discharged as waste media fromthe cell bed by flow through the plurality of apertures 48, as shown byarrows in FIG. 7.

Because of the radially outward flow of the perfusing liquid and thearrangement of the outlet apertures 48, the cells within the cellculture bed are uniformly perfused with nutrients. For example, theradial flow of the perfusing liquid to a plurality of equiangularlyspaced outlet apertures promotes a uniform fluid flow from the inlet,and over the cell bed to the perimeter outlet locations on thecircumference of the cell bed.

The base 30 has apertures 32 and 34 through which the ports 44 and 46can extend in a fluid tight manner, for example, via seals (not shown)between the apertures and the ports. Alternatively, the apertures arenot required if port 44 extends upwardly through the centerport 28 andport 46 extends directly from the base (not shown). The base alsoincludes a generally centrally located outlet port 36 for the wasteliquid displaced from the biochamber. The waste liquid from theapertures 48 flows radially inward, through the space between the bottomsurface of the disc 40 and the top surface of the base 30, to the port36 and is thereby discharged from the biochamber. The port 36 may beco-axial, but can also be slightly offset from the radial center of thebase 30 in order to accommodate the aperture 32 for the media inlet port44. Alternatively, the aperture is not required if port 44 extendsupwardly through the centerport 28 (not shown).

The biochamber top 20 is secured to the base 30 in a fluid tightfashion, with the membrane 50 therebetween, as mentioned above. Aconcentric labyrinth path of ribs may be included which extend inwardlyfrom the top 20 to support the membrane 50 against distortion due to thefluid pressure of the perfusion liquid in the cell bed. The ribs 22maintain a precise spacing between the top surface of the disc 40 andthe bottom surface of the membrane 50, i.e., a precise thickness for thecell growth bed. This thickness may be about 4 mm in order to assureadequate oxygenization of the cells within the cell growth bed.Alternatively, a series of periodic supports extending downwardly fromthe top 20 and a thin porous disc to which the membrane 40 is laminatedmay used to maintain the position of the membrane to provide a precisethickness for the cell growth bed (not shown).

The ribs 22 also form a labyrinth-like gas chamber through which anoxygenization fluid, such as air, can flow to supply oxygen which isdiffused through the membrane and into the cell bed. The two ends of thelabyrinth may be adjacent one another so that the oxygenizing air can besupplied to the gas inlet port 24 and discharged through the gas outletport 26. Alternatively, if periodic supports have been used instead ofthe concentric labyrinth path of ribs, the gas inlet port and outletport can be located near the perimeter of the top 180° opposite to eachother so that oxygenizing air can be supplied to the gas inlet port anddischarged through the gas outlet port (not shown).

A bell-like raised center port 28 is formed at a radial center of thetop 20 and forms a chamber sealed by the annular rib 29 bearing againstthe membrane. Cells can be inoculated into the cell growth chamber via acenter port septum 28A. For this, a non-latex needle septm mau besecured to a port feature with an air-tight band for acsess directly tothe cell residence area. Alternatively, a tubing line can extend fromthe center port that may be connected to an external container of cellsusing a sterile tube welder (not shown).

Referring to the detailed illustrations of the biochamber top, base andcell bed disc shown in FIGS. 8 through 10, the biochamber top 20 isshown in FIGS. 8A and 8B. The top 20 is preferably formed of aninjection molded transparent, non-reactive plastic such as polystyreneor PETG. It has a generally disc-like main portion 20A bounded at itsradially outer periphery by a flange 20B. The flange 20B has an equallyspaced plurality of bolt holes 20C, through which may pass bolts (notshown) for securing the top 20 to the base 30. Alternatively, an EMA(Electro-magnetic) weld can be used for securing the top to the base(not shown).

The raised center port 28 has a generally bell shape and a centralseptum 28A. The septum is a gas and liquid impermeable barrier that maybe pierced by an injection needle and is self-sealing when withdrawn.Alternatively, a tubing line can extend from the center port that may beconnected to an external container using a sterile tube welder (notshown). An equally spaced plurality of radial reinforcing ribs 20Dextend from the main portion 20A between the center port 28 and anannular reinforcing rib 20E adjacent the rim 20B.

The ribs 22 are generally annular in orientation and form a labyrinth20F as shown by dash lines in FIG. 8A. The labyrinth may be convolutedsuch that an oxygenizing gas is able to flow through a gas chamberdefined thereby over the entire cell growth bed. The opposite ends ofthe labyrinth are preferably adjacent one another at the radially outerend of the main portion 20A. An inlet port 24 and the outlet port 26communicate with the opposite ends of the labyrinth. Alternatively, ifperiodic supports have been used instead of the concentric labyrinthpath of ribs, the gas inlet port and outlet port can be located near theperimeter of the top 180° opposite to each other so that oxygenizing aircan be supplied to the gas inlet port and discharged through the gasoutlet port (not shown).

A radially innermost rib 29 may be a continuous annulus which, incooperation with the membrane 50, seals the gas chamber defined by thelabyrinth from the interior of the central port 28. A radially outermostcontinuous rib 20F defines the outermost limit of the labyrinth. Thetips of all of the ribs 20F, 22 and 29 are coplanar with the bottomsurface of the flange 20B so that the ribs seal with respect to themembrane 50 when the biochamber is assembled. Alternatively, if periodicsupports have been used instead of the concentric labyrinth path ofribs, the tips of all the supports are coplanar with the bottom surfaceof the flange 20B so that the position of the membrane 50 is controlled(not shown).

Referring to FIGS. 9A and 9B, the cell bed disc 40 is preferably alsoformed of an injection molded transparent, non-reactive plastic such aspolystyrene or PETG. It has a generally disc-like main portion 40Abounded at its radially outer periphery by the annular lip 42. The uppersurface of the main portion is generally smooth and unobstructed andforms a surface of adhesion for the cell colony being cultured.

Referring to FIGS. 10A and 10B, the base 30 is preferably also formed ofan injection molded transparent, non-reactive plastic such aspolystyrene or PETG. It also has a disk-like main portion 30A bounded bya raised peripheral flange 30A having an upper surface 30B and boltholes 30C. Alternatively, the bolt holes are replaced with features toperform an EMA weld to secure the base to the top (not shown). Whenassembled, the disc 40 fits entirely within the bounds of the peripheralflange 30A with the surface A closely adjacent, and coplanar with, thesurface 30B. A peripheral lip 30D extends upward from the radially outeredge of the flange 30B to position and retain the membrane 50 duringassembly of the biochamber.

The upper surface of the main portion 30A has a plurality of raisedregions 30E which support the bottom surface of the disc 40 and maintaina separation between disc 40 and base 30, thereby defining the fluidpath for the return flow of the waste media to the central outlet port34. Recesses 30F and 30G surround each of the apertures 32 and 34, andcan house resilient elements for sealing the apertures. Alternatively,the apertures are not required if port 44 extends upwardly through thecenterport 28 and port 46 extends directly from the base (not shown).

A plurality of radial supporting ribs 30H extend from the bottom surfaceof the base and extend between the annular supporting ribs 30I and 30J.Annular reinforcing enlargements 30K, 30L and 30M surround the apertures32, 34 and 36, respectively.

In assembling the biochamber, appropriate seals are positioned at theapertures 32 and 34, and the disc 40 is positioned within the base 30with the nipples of the ports 44 and 46 sealingly extending through therespective apertures as shown schematically in FIG. 7. Alternatively,the apertures and related seals are not required if port 44 extendsupwardly through the centerport 28 and port 46 extends directly from thebase (not shown). The membrane 50 is then placed over the disc 40 andthe flange 30A, and is held within the lip 30D. Alternatively, themembrane 50 is laminated to a porous disc to provide additionalmechanical stability prior to placement over the disc 40 (not shown).The top 20 is then placed over the base with the bolt holes 20C and 30Cin alignment, and bolts are passed through the bolt holes and tightenedto unify the biochamber. Alternatively, an EMA weld is used to join thetop to the base (not shown). At this time, the outer annular rib 20Fwill clamp the membrane 50 against the radially innermost portion of thesurface 30B to seal the interior of the biochamber.

Referring to the schematic FIGS. 5 and 6, the biochamber 10 is heldwithin a casing of a cell cassette 100 and forms a preassembled,disposable unit. The biochamber is secured to a cassette base 60 of thecassette casing. In the illustrated system, the base 60 has a supportflange 62 with a central aperture having a plurality of holes. The boltsused to secure the top to the base of the biochamber 10 can also extendthrough these holes for securement of the biochamber to the cassettebase 60. Alternatively, mounting clips can be mounted to the base forthe securement of the biochamber to the base at 3 or more equally spaceperimeter positions (not shown). The cassette base is closed from aboveby a top 64 and from below by a tray 66, and a media supply container 68is mounted to a front surface of the cassette base 60 for supplying agrowth media to the biochamber. The media supply container 68 isprovided with a media supply line 68A connected to the media inlet port44 of the biochamber. Pressurized air from an air pump is supplied tothe air space above the media in the media supply container via the line68B, and in this way the growth media is pressurized so as to provide aconstant flow rate of media to the cell growth bed 25 in the biochamber.Additional growth media is supplied to the container via the mediacontainer supply conduit 68C.

A cell harvest bag 70 (or other cell harvest collection device) may beconnected to the harvest port 46 of the biochamber via the conduit 72and the harvest valve 74.

A waste liquid bag 76 is positioned below the biochamber and rests onthe tray 66 of the cell cassette. It receives waste liquid from thebiochamber via a drip chamber device 78 attached to a valve plate 80 atthe rear of the cell cassette. The drip chamber includes a siphon breakto permit a precisely controlled low pressure within the biochamber. Adrip counter (not shown) can be associated with the drip chamber device.It counts the drops of waste liquid to detect the flow rate.

The waste liquid reaches the drip chamber device via a waste valve 82.Gas pressure at the center port 28 of the biochamber is used to regulateflow through the drip chamber, via the center port valve 84 and the line84A.

Also attached to the valve plate may be an air pump supply port 86 forsupplying compressed air at a constant pressure to the media supplycontainer via the air pump supply line 68B; gas in and out ports 88 and90 for supplying fresh oxygenizing gas to, and discharging spentoxygenizing gas from, the biochamber; a media supply valve 92 connectedto the media container delivery line 68A; HBSS valve 94; and trypsinvalve 96. Alternatively, valve 94 and valve 96 are replaced with asingle valve for addition of a range of reagents, such as HBSS andtrypsin (not shown).

Each cassette may also include a “key” containing a non-volatile memorydevice and a clock. Before use, the key of the cassette is initializedwith tracking data, protocol commands and real time via the systemmanager 300. The key is used by the system electronics during the cellproduction process to record pertinent data as well as to access theprotocol commands.

In use, a sterile, single use disposable cell cassette 100 is suppliedin a protective package. It includes the medium supply reservoir 68,medium flow control (not shown), the biochamber 10, the waste reservoir76, the harvest reservoir 70, a key and the necessary plumbing, valvingand packaging to interconnect and support the components. Alternatively,the harvest reservoir can be provided as a separate component and thenconnected to the cassette at the time of harvest using a sterile tubewelder.

In operation, the key is first initialized by the system manager. Oncethe key has been initialized, it is transferred to the processor 400.The processor includes a multiaxis gyrator (“wobbulator”) 410. Thewobbulator includes a support table 412 onto which the cassette can besecured. The wobbulator has mechanical linkages 414 for pivoting thesupport table 412 about two orthogonal horizontal axes.

When a cell cassette is loaded in the processor 400 and clamped onto thesupport table 410, and the key indicates that inoculation is required,the processor provides an automatic sequence of inoculation operations.For example, the inoculation sequence can consist of the followingsteps. First, the wobbulator table 412 is brought to a horizontal homeposition. The cell cassette 100 is then primed with growth media to therequired volume, employing gravity feed of media from the reservoir 68.Alternatively, pressure can be applied to the reservoir to facilitatetransfer of media. During this time, the harvest valve 74 is closed andthe waste valve 82 is open so that media in the biochamber 25 can flowthrough the apertures 48 but not through the harvest port 46. The cellcassette is then tilted to generate a bubble to be used in distributingthe cells. The biochamber is then inoculated with cells. This may bedone via a hypodermic syringe passing through the center port septum andthe membrane. Alternatively, a container with cells can be connected toa tubing line extending from the centerport. using a sterile tube welderand then cells inoculated using gravity or pressure (not shown). Thewobbulator then oscillates the cell cassette (i.e., agitates thecontents therein) according to a predetermined program to distribute thecells on the upper surface of the disc 40. At this time, the bubble aidsin the even distribution of the cells within the biochamber. Remainingair is then purged through the center port and the cassette is removedfrom the processor. The cassette is then ready for incubation.

The biochamber 10 may be substantially filled (preferably completelyfilled) with the growth media, which may be displaced by cells duringinoculation. For example, the biochamber may be filled to around 80%total volume with the growth media. The cells, during inoculation, maybe suspended in the same growth media, or a different liquid/media.During inoculation, the biochamber may be less than completely filled(e.g., 90% total volume), so that cells may be distributed evenlythroughout the interior of the biochamber during agitation. Afterinoculation, preferably, the biochamber is substantially filled(preferably completely filled).

The cassette is then placed in the incubator 200 where the biochamber ismaintained in a horizontal position to allow cells to settle by gravityonto the bottom surface of the biochamber where they remain throughoutculture. The incubator is an instrument capable of accepting cellcassettes for cell production. It can take the form of a rack 210 towhich plural cassettes are attached. It mates with the cassette toprovide control over the culture environment within the cassette. It isalso connected to the system manager 300 and to the key for storing theincubation start time and date on the key, and incubation data iscontinually provided to the key during the incubation sequence. The keyalso receives information on abnormal events, such as alarms or powerfailures, the amount of medium used and the incubator identification.The incubator controls the flow of medium through the growth chamber,the temperature of the growth medium reservoir 68, and the concentrationand flow rate of gases delivered to the gas chamber in the biochamber,based on control settings stored in the key. The incubator also monitorsvarious safety/alarm parameters to assure that the cell productionprocess is proceeding as expected. This can be done for a number ofincubators through the system manager computer or by use of anindependent incubator computer.

Following the completion of culture, the cassette is removed from theincubator and placed back into the processor where the harvest washprocess previously described is performed.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLES Example 1 Tissue Repair Cell (TRC) Culture and Wash TechniqueProtocols TRC Culture

Fresh bone marrow mononuclear cells (BM MNC) that were isolated byFICOLL® from the blood of normal donors were purchased from PoieticsInc. (Gaithersburg, Md.). Alternatively aspirated bone marrow (BM) wasreceived as a clinical specimen from patients and separated on FICOLL®to create a mononuclear cell prep. Cell concentration was assessed usingan automated cell counter, and BM MNC were cultured by the method ofLundell, et al., described above. Prior to inoculation, culture chamberswere primed with culture medium consisting of IMDM, 10% fetal bovineserum, 10% horse serum, 5 μM hydrocortisone, and 4 mM L-glutamine.Medium was passed through the culture chambers at a controlled, rampedperfusion schedule during the 12 day culture process. The cultures weremaintained at 37° C. with 5% CO₂ and 20% O₂.

CYTOMATE® Wash Method

CYTOMATE® is a fully automated system designed for washing andconcentrating white blood cell products. It includes anelectro-mechanical instrument and single-use pre-sterilized disposablesets that provide a wash circuit for each batch of cells to beprocessed. It incorporates spinning membrane technology that provides atangential flow affect to prevent excessive filter loading with cells.

Setup

1. Load wash circuit set onto CYTOMATE® instrument

2. Connect bags:

-   -   Bag of cells harvested from culture process (TRCs in 800 to 1000        mL volume of culture process fluids, e.g. culture medium,        harvest enzyme solution).    -   Buffer solution (2000 to 3000 mL, Isolyte supplemented with 0.5%        HSA).    -   Collection bag for washed cells (120 mL to 180 mL collect        volume).    -   Supernatant bag (2600 to 3900 mL waste liquid not collected with        cells).

CYTOMATE® Procedure:

1) Prime wash circuit with buffer solution and initiate recirculation

2) Transfer cells from harvest bag into wash circuit, reduce liquidvolume by recirculating cells through wash circuit while removing liquidthrough filter in wash circuit (filter is spinning to provide tangentialflow affect and prevent filter clogging). Removed liquid is collected insupernatant bag.

3) Use buffer to rinse residual cells from harvest bag into washcircuit.

4) Wash cells by recirculating cells through wash circuit,simultaneously removing liquid through the spinning filter intosupernatant bag and adding buffer solution as required to maintainvolume.

5) Transfer washed cells into collection bag.

6) Use buffer to rinse residual cells from wash circuit into collectionbag.

Wash Harvest Method

The wash-harvest process begins by displacing culture media from aculture chamber with a biocompatible rinse solution (Step 1, Table 8,above). The rinse solution (normal saline or other isotonic solution) isthen replaced with a cell harvest enzyme solution (Step 2, Table 8),with porcine trypsin being used the majority of the time. Othernon-animal derived harvest reagents such as TRYPLE™ (Invitrogen,Carlsbad, Calif.) and TRYPZEAN™ (Sigma, St. Loius, Mo.) have also beenused successfully. The culture chamber is left to incubate for a periodof time (5-60 minutes, preferably 15 minutes with porcine trypsin) asthe enzyme works to dissociate the cells from each other and from theculture surface (Step 3, Table 8). When the enzyme incubation iscomplete, a second, typically injectable grade, rinse solution (Isolyteor Normasol) displaces the enzyme solution (Step 4, Table 8). At thispoint the chamber contains the detached cells, which remain settled onthe cell surface, and is suspended in an injectable-grade solution. Inorder to increase the final harvested cell concentration and reduce thefinal volume, a portion of this rinse solution is displaced with air(Step 5, Table 8). When the final liquid volume (100-350 ml) is achievedin the culture chamber, the chamber is agitated in order to bring thesettled cells into solution (Step 6, Table 8). This cell suspension isdrained into a cell collection container (Step 7, Table 8). Anadditional amount of the injectable-grade solution may be added to thecell culture chamber and a second agitation could occur in order torinse out residual cells if necessary (Steps 8 & 9, Table 8). This finalrinse is then added to the cell collection container (Step 10, Table 8).

Comparision of Cytomate vs Wash Harvest

On the day of harvest the TRCs were split into two cultures. The firstculture was harvested and concentrated per the standard CYTOMATE®process. The TRCs in the first culture were harvested by trypsinization(0.025% trypsin in 0.9% sodium chloride), and washed to deplete culturematerials using a CYTOMATE® (Baxter International, Inc.) cell processorper manufacturer's instructions. The cell product was washed with apharmaceutical grade electrolyte solution supplemented with 0.5% HSAinto a 150 mL volume and used, as is, or concentrated to 15 ml or 5 mLvolumes in a biocompatible bag. The second culture of TRCs was harvestedusing a draft of the combined wash-harvest ARS Processor Sequence, and amodified concentration process designed to further reduce residuals withan additional dilution step.

To create a concentrated TRC suspension, the collected cells from eachwash were centrifuged to a 20 ml volume and transferred to a smaller bagsuch as a Cryocyte bag. Once in the final container a secondcentrifugation step is performed to concentrate to a final volumebetween 4.7 and 20 mls, creating a dose of cells ranging from 35-300million cells in 6 ml⁺/−1.3 ml, but up to 20 ml of injectable gradesolution depending on the final application.

Example 2 The Wash Harvest Increased TRC Quality Over the CYTOMATE® Wash

TRCs isolated using the Wash Harvest had greater cell viability, greatercell yield, less residual BSA, higher total numbers of progenitor cell,stem cell, immune cell and endothelial cell markers, increased abilityto form colonies, comparable viability after needle passage, higherlevels of anti-inflammatory cytokines and higher levels of indoleamine2,3-dioxygenase (IDO). These improvements in the TRC population due touse of the novel Wash Harvest process allows the population to be usedas a more effective tissue repair therapeutic agent when compared tocurrent state of the art processes.

Materials and Methods

Cell Count/Viability

Cell count and viability were measured by Nucleocounter or trypan blueexclusion. The manufacturer's protocol was used for cell counting usingthe Nucleocounter. Briefly, the cell suspension was diluted to between100,000 and 10,000,000 cells/ml, and a nucleocassette aspirates the cellsuspension. The necleocassette is placed into the Nucleocounter forautomated propidium iodide staining, including cell count and viability.Where Nucleocounter data was not available, trypan blue exclusion andhemocytometer (manual counting) was used to enumerate cell number andviability. Over the course of 27 sample analyses, the Nucleocounter cellcounts were within 13% of the Trypan Blue counts and viability waswithin 4%.

During these experiments the product sampling at the post-cultureprocessing phases varied depending on the assays and other uses for thecells. The following strategy was used in order to get the most accuratetotal viable cell numbers from the data. The total cell number of eachsample taken was calculated, and then that number was multiplied by theviability of the next processing step and then added to the viable cellcount at that step. For example, if a sample of 10×10⁶ total cells wasremoved from the washed product and the viability of the cells afterconcentration was 80%, then 8 ×10⁶ viable cells were added to the totalviable count of the concentrated product, to represent what would havebeen there had the non-standard sampling not occurred. Once thenon-sampled totals were calculated, the true manufacturing sample volumeof 29 mL was subtracted from each washed product total cell number.

Residual Levels

Supernatant from the final TRC concentration process for each experimentwas used to measure the level of residual BSA (via ELISA)and Trypticactivity (via the Quanticleave assay). A BSA ELISA assay was used tomeasure and compare the levels of residual BSA from the culture medium.

Concentration Protocol

To create a concentrated suspension, the collected cells are centrifugedto a 20 ml volume and transferred to a smaller bag such as a Cryocytebag. Once in the final container a second centrifugation step isperformed to concentrate to a final volume between 4.7 and 20 mls,creating a dose of cells ranging from 35-300 million cells in 6 ml+/−1.3 ml, but up to 20 ml of injectable grade solution depending on thefinal application. Preferred volumes for the concentrated cultures are 6ml +/−1.3 ml. When TRCs are retrieved from storage, cultures were thawedin a 37° C. circulating water bath.

Cell, Viability and % CD90⁺

Cell viability and % CD90⁺ cells were measured by flow cytometry. Cellswere washed and resuspended in 1× Dulbecco's phosphate buffered saline(PBS; Gibco) containing 1% bovine serum albumin. Tubes containing 10⁶cells in 0.5 ml were stained on ice with various combinations offluorescently-conjugated monoclonal antibodies. Viability was determinedby 7-Amino-Actinomycin D (7AAD) (Beckman Coulter). 7AAD only entersmembrane-compromised cells and binds to DNA. Cells were stained withPC5-conjugated anti-CD90 (Thy1) antibodies and FITC-conjugated anti-CD14(Beckman Coulter). After 15 minutes, cells were washed and resuspendedin 0.5 ml PBS/BSA for analysis on the Epics XL-MCL (Beckman Coulter)flow cytometer.

Intracellular Cytokine Analysis by Flow Cytometry

Cytokine expression by TRCs produced using the Wash Harvest process wasdetermined quantitatively by 2-color intracellular flow cytometricanalysis. Briefly, TRCs were incubated overnight with or withoutbacterial lipopolysaccharide (LPS) in the presence of brefeldin A toenhance intracellular cytokine accumulation in the Golgi apparatus whileblocking cytokine secretion. The TRCs were stained for cell surfacemarkers by incubation with FITC or Cy5PE-conjugated monoclonalantibodies (mAbs) (anti-CD14, CD66b, CD90 or control mAbs). Thelymphocyte subpopulation was defined by gating on cell size based onforward (FSC) and granularity based on side (90°) light scatter (SSC).Subsequently, the cells were fixed using paraformaldehyde andpermeabilized in saponin prior to staining with cytokine-specificPE-conjugated monoclonal antibodies (IL-6, IL-10, IL-12 or irrelevantcontrol) as indicated in the left column of Table 4. Data for 2-coloranalysis was acquired on a Becton Dickinson FC500 flow cytometer.

CFU Frequency Analysis

For colony forming unit-fibroblast (CFU-F) assays, cells were plated in1 ml LTBMC in 35 mm tissue culture treated dishes. For TRCs, 500 and1,000 cells were plated per dish. Cultures were maintained for 8 days at37° C. in a fully humidified atmosphere of 5% CO₂ in air. CFU-F colonieswere then stained with Wright-Giemsa and colonies with greater than 20cells were counted as CFU-F.

For colony forming unit-granulocyte/macrophage (CFU-GM) assays, cellswere inoculated in colony assay medium containing 0.9% methylcellulose(Sigma), 30% FBS, 1% BSA, 100 μM 2-mercaptoethanol (Sigma), 2 mML-glutamine (Gibco), 5 ng/ml PIXY321, 5 ng/ml G-CSF (Amgen), and 10 U/mlEpo. TRCs were plated at 1,500 and 3,000 cells per ml. Cultures weremaintained for 14 days and were colonies greater than 50 cells werecounted as CFU-GM.

Cell Delivery Through Needles

To test the effects of needle delivery on TRC cell counts and viability,samples was run from five of the Wash Harvest products and three of theCYTOMATE® wash products. These delivery experiments tested the abilityof stored cells to be delivered to patients without loss of viability orconcentration after passing through 25 gauge needles.

After 24 hours storage at 4° C., cryocyte bags containing TRCs wereremoved from refrigerator and massaged to resuspend and homogenize theTRCs for sampling. Two 0.5 mL samples were collected via 3 mL syringeand placed into tubes labeled as “TRC.” Afterwards, two additional 3 mLsyringes were placed onto the 3 way-stopcock valve and an additional 0.5mL per syringe was taken. Twenty-five gauge needles (1½ inch, BD) werethen screwed onto these additional 2 syringes (3 mL) and designated as“TRC, 25 gauge, 1½ inch needle.” Afterwards, the remaining TRC sampleswere measured and volume recorded. Onto these 3 mL syringes, 25 gaugeneedles (1½ inch) were added to two syringes and 25 gauge needles (3inch, spinal needle) added to other two syringes. The entire 0.5mlneedle samples within the 3 mL syringes with needles were dispensedusing a syringe pump (Harvard Apparatus, Holliston Mass.) at a rate of2.5 mL per minute or 0.5 mL in 12 seconds into polystyrene round bottomtubes. After cell delivery through needles, all samples were evaluatedfor cell counts using nucleocounter.

Western Analysis of DO Expression

TRCs express an inducible immunoregulatory enzyme designated indoleamine2,3 dioxygenase (IDO) which is associated with down-regulation ofinflammatory responses. Tissue Repair Cells (TRCs) were derived usingthe new wash-harvest process as described in the current invention.After harvest, TRCs were incubated for 24 hours in medium alone ormedium containing 1000 units/ml recombinant human interferon-γ (IFN-γ).Protein extracts from total cell lysates were separated on a 10% SDSpolyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF)membrane and probed using a mouse anti-human DO-specific monoclonalantibody. A goat anti-mouse horse-radish peroxidase conjugatedsecond-step antibody was used for subsequent visualization bychemiluminescence. This experiment demonstrates a characteristic 44kilodalton (kd) band corresponding specifically to expression of IDOprotein by TRCs after induction with IFN-γ.

Results

Total Viable Cell Count

FIG. 11 shows that the Wash Harvest repeatedly produced higher numbersof total viable cells post-wash than when the CYTOMATE® wash was used,after the manufacturing sample of 29 mL was subtracted. The datasummarized in FIG. 11 was from 9 productions that compared the CYTOMATE®wash to the hybridization wash from the same donor. Aastrom HarvestReagent (porcine trypsin) was used in all nine of these productions. Theaverage post-wash total viable cell yield for the CYTOMATE® wash was66.5×10⁶±36.8 ×10⁶ viable cells while the average post-wash total viablecell yield for the hybridization wash was 144×10⁶±50.9×10⁶ viable cells.Variability in total yield appears to be donor-dependent.

Viability

FIG. 11 also shows that the Wash Harvest repeatedly produced higher cellviability post-wash than when the CYTOMATE® wash was used. The percentviability was performed on cells isolated from the same 9 donors asdescribed above. The wash-harvest product shows a more consistentviability with a standard deviation of 2%, compared with the CYTOMATE®wash viability standard deviation of 10%.

Count and Viability After 24 Hour Storage

FIG. 11 also shows that the Wash Harvest repeatedly produced higher cellviability, even after 24 hours of storage at 4° C., than when theCYTOMATE® wash was used. The percent viability after 24 hours of 4° C.storage.was measured from cells from 5 donors.

BSA Concentration Post-Wash

FIG. 11 also shows that the Wash Harvest produced lower concentrationsof the residual BSA left over from the culturing of the TRCs than whenthe CYTOMATE® wash was used. Low BSA levels are necessary in order togenerate a pharmaceutical product appropriate for administration tohumans.

Cell, Viability and % CD90⁺ Via Flow

FIG. 11 shows that the Wash Harvest repeatedly produced higherpercentages of CD90⁺ TRCs than when the CYTOMATE® wash was used inunconcentrated cells. CD90⁺ cells represent bone marrow stromal cellswhich have stem/progenitor cell properties and are useful for repairingvarious tissue types.

FIG. 12 shows that the Wash Harvest repeatedly produced higher totalnumbers of viable CD90⁺ TRCs than when the CYTOMATE® wash was used. Theaverage total viable CD90⁺ cells in the wash-harvest final product was42.4×10⁶±16.5 ×10⁶. The average total viable CD90⁺ cells in theCYTOMATE® wash final product was 19.1×10⁶±10.8 ×10⁶.

CD 14⁺ Auto⁺%

FIGS. 12 and 13 shows that the Wash Harvest repeatedly produced higherpercentages and total numbers of viable CD14⁺ Auto⁺ TRCs than when theCYTOMATE® wash was used. The average total viable CD14⁺ Auto⁺ cells inthe new wash concentrated product over these six donors is 34.8×10⁶±9.08×10⁶. The average total viable CD 14⁺ Auto⁺ cells in the CYTOMATE® washconcentrated product over these six donors is 16.5×10⁶±5.37 ×10⁶.

VEGFR1⁺%

FIGS. 12 and 13 shows that the Wash Harvest repeatedly produced highertotal numbers and percentages of VEGFR1⁺ TRCs than when the CYTOMATE®wash was used in concentrated cells, demonstrating that more endothelialcells are in the final mixture. In each of the five experiments wherethis was measured, more viable VEGF-R1⁺ cells were seen in the WashHarvest product compared to the CYTOMATE® control. The average totalviable VEGF R1⁺ cells in the CYTOMATE® wash concentrated product overthese five donors is 16.5×10⁶±5.37 ×10⁶.

CFU-F Frequency

FIGS. 12 and 14 shows that the Wash Harvest process produced comparableCFU-F frequencies compared to the CYTOMATE® wash. The average of theCFU-F frequency ratio was 1.03.

FIGS. 13 and 15 shows that the Wash Harvest produced greater numbers ofCFU-F per dose than the CYTOMATE® wash. The total CFU-Fs per dose wascalculated by multiplying the frequency of CFU-Fs per cell by thepost-wash total viable cell count. The average CFU-Fs per dose for thenew wash process across these 8 experiments is 7.26×10⁶±5.22×10⁶. Theaverage CFU-Fs per dose for the CYTOMATE® wash process across these 8experiments is 3.01×10⁶±1.37×10⁶.

CFU-GM Frequency

FIGS. 12 and 16 shows that the Wash Harvest, in almost every case,produced equal or higher frequency of CFU-GM per dose than the CYTOMATE®wash. The average of the CFU-GM frequency ratio is 1.37.

FIGS. 13 and 17 shows that the Wash Harvest, in almost every case,produced greater total numbers of CFU-GM per dose than the CYTOMATE®wash. The total CFU-GMs per dose was calculated by multiplying thefrequency of CFU-GMs per cell by the post-wash total viable cell count.The average CFU-GMs per dose for the new wash process across these 6experiments was 0.42×10⁶±0.19×10⁶. The average CFU-GMs per dose for theCYTOMATE® wash process across these 6 experiments was 0.17×10⁶±0.11×10⁶.

Needle Delivery

FIG. 17 shows that the cell viability of the Wash Harvest product doesnot show substantial change after delivery through 25 and 30 gaugeneedles. FIG. 18 shows similar data from CYTOMATE® washed products.

CFU-Fs were tested on post-needle delivery cells from experimentQTRC107000021. FIG. 19 shows the CFU-Fs per 100 cells for the threeconditions. TRCs from the wash-harvest had slightly higher viabilitythan TRCs from the CYTOMATE® wash.

This data demonstrates comparability between the processes on theability to deliver the cells via needles at the end of processingwithout losing substantial cell viability following transit throughsmall gauge needles.

Cytokine Secretion

The cytokine secretion profile by the TRCs from the CYTOMATE® wash iscomparable on a per cell basis on almost all cytokines evaluated for thewash-harvest. However, on a unit dose basis (all cells coming out ofprocess), the,total cytokine secretion per dose is generally higher fromthe wash-harvest (FIG. 20), thus as a concentrated composition the cellpopulation is much more functional than the previous population.

TABLE 9 Flow cytometric analysis of TRCs and TRC-subpopulations forintra-cellular cytokine expression. TRC SUBPOPULATIONS TRCs CD14⁺Auto⁺CD14⁺Auto⁻ CD66b⁺ CD90⁺ Lymphocyte LPS: − + − + − + − + − + − + Control:0.4 0.3 IL-6: 11 15 6.2 9.2 0.9 1.2 0.6 0.7 1.6 1.1 1.1 1.3 IL-10: 6.47.9 2.8 4.5 0.4 0.6 0.5 0.4 0.9 0.6 0.1 0.1 IL-12: 0.0 0.0 0.3 0.2 0.10.1 0.1 0.1 0.0 0.0 0.0 0.0

Intracellular flow cytometric analysis to evaluate the frequency ofcells producing IL-6, IL-10 and IL-12 was performed on TRCs producedusing wash-harvest. The mean percentage of cytokine-positive cells forN=2 experiments is shown in Table 9. These observations demonstrate thata significant frequency (6-15%) of TRCs produce either IL-6 or IL-10. Incontrast, intracellular IL-12 production by TRCs or TRC subpopulationswas not detectable above background levels of staining observed forirrelevant control antibodies. IL-12 was not detectable above backgroundlevels regardless of stimulation with inflammatory mediators such asbacterial lipopolysaccharide (LPS). Overall, these data are highlyconsistent with the cytokine secretion profiles defined by Luminex andELISA analysis of TRC supernatants (FIG. 20) which demonstrate highsecretion of multiple angiogenic and immunomodulatory cytokines in thecomplete absence of detectable IL-12, a central pro-inflammatorymediator.

IDO Expression

Indoleamine 2,3 dioxygenase or IDO is an immunoregulatory enzyme. TRCsproduce higher levels of IDO mRNA in response to exposure to IFNγ (FIG.24). TRCs also produce higher quantities of IDO protein in response toexposure to IFNγ (FIGS. 25).

PDL1 Expression

TRCs express high levels of PD-L1 in response to inflammatory induction(FIG. 31). TRCs were incubated without (un-induced) or with (induced)1000 units per ml of interferon-γ for 24 hours prior to staining withfluorochrome-conjugated isotype control, or anti-PD-L1 monoclonalantibodies for flow cytometric analysis. These observations demonstratethat TRCs up-regulate PD-L1 (>75% expression), a key inhibitory receptorimplicated in down-regulation of immune and inflammatory responses.

Summary

The Wash Harvest creates a healthier cell product (p<0.05) at each stage(post-wash, post-concentration, and after 24 hours storage), as well aslower residual serum proteins (p<0.05) when compared to the CYTOMATE®wash (FIG. 11). There are no statistical differences in the percentagesof CD90⁺, CD14⁺ auto⁺, or VegFR1⁺cells, or in the F or GM colony formingcapabilities. Therefore, the cell product is comparable in profile butmuch healthier and purer.

TABLE 10 Statistics for FIG. 11. Donor Paired Ratio New/Cytomate StdevCurrent stdev new stdev p value <0.05 % Viability post-wash 1.32 0.1971.6% 9.7% 92.8% 2.2% X (n = 9) % Viability post- 1.24 0.11 73.7% 6.2%91.2% 3.7% X concentration (n = 9) % Viability post-24 hr 1.32 0.3366.8% 14.2% 84.5% 3.8% X storage (n = 5) % CD90⁺ in final 1.07 0.2028.8% 13.9% 30.1% 13.4% product (n = 7) % CD14auto⁺ in final 1.24 0.1026.0% 6.6% 31.7% 6.4% product (n = 6) % VegfR1⁺ in final 1.24 0.21 32.2%10.7% 38.5% 8.6% product (n = 5) CFU-F frequency, final 0.95 0.25 7.123.60 7.07 4.17 product (n = 3) CFU-GM frequency, 1.43 0.65 0.22 0.120.27 0.10 final product (n = 3) Residual BSA, ug/ml 0.61 0.27 2.65 0.761.51 0.56 X (n = 11)

When looking at the total cells in the final product (the final dosethat comes out of the process), there are statistically greater numbersof total viable cells, total viable CD90⁺ and CD14⁺auto⁺ (two cellssecreting the Immunomodulatory cytokines).

TABLE 11 Statistics for FIG. 12. Donor Paired Ratio Current p valueNew/Cytomate Stdev (average) stdev New (average) stdev <0.05 Totalviable 1.91 0.72 95.0E⁺6 47.5E⁺6 161.1E⁺6  57.3E⁺6 X cells post- wash (n= 9) Total viable 2.42 1.17 58.3E⁺6 32.2E⁺6 113.9E⁺6  38.6E⁺6 X cellsfinal product (n = 9) Total viable 1.98 0.49 18.8E⁺6  8.9E⁺6 34.6E⁺610.5E⁺6 X CD90⁺ in final product (n = 7) Total viable 2.16 0.40 20.4E⁺6 5.4E⁺6 43.2E⁺6 11.7E⁺6 X CD14auto⁺ in final product (n = 6) Totalviable 2.12 0.40 26.0E⁺6 13.1E⁺6 53.9E⁺6 26.4E⁺6 VegfR1⁺ in finalproduct (n = 5) CFU-F per 1.71 0.68  5.8E⁺6  3.8E⁺6  8.9E⁺6  4.1E⁺6dose, final product (n = 3) CFU-GM per 2.64 1.46 dose, final product (n= 3)

The Wash Harvest produced TRCs with higher viability and lower residuallevels from cell culture. This allows greater numbers of TRCs to beharvested from each culture.

Further, the Wash Harvest produced TRCs with increased percentages ofCD90⁺, CD14⁺ Auto⁺ and VEGFR1⁺ cells when compared to TRCs isolatedusing the CYTOMATE® wash. Also the wash-harvest produced TRCs thatsecreted more anti-inflammatory cytokines pre cell dose includingIL-1ra, IL-10 and IL-6 and proteins with anti-inflammatory effects likeIDO and PDL1. Further, TRCs do not express pivotal pro-inflammatorycytokines like IL-12. This shows that the TRCs isolated by the WashHarvest method have greater tissue repair and anti-inflammatorypotential because of the higher percentage of bone marrow stromal cells,endothelial cells, and monocyte/macrophage cells.

Also, the Wash Harvest technique consistently produced higher CFUs thanthe CYTOMATE® wash technique. This shows that the TRCs produced by theWash Harvest technique have a greater number of progenitor and stemcells than TRCs produced by the CYTOMATE® wash technique.

Moreover, the Wash Harvest produces TRCs that do not lose substantialviability when passed through a 25 or 30 gauge needle, which may benecessary for therapeutic administration. The Wash Harvest TRCsperformed about the same, if not slightly better than TRCs producedusing the CYTOMATE® wash.

Example 3 Enhanced Bone Repair Potential of TRCs from Wash-Harvest Basedon Increased Numbers of CD90⁺ Cells

The bone-forming or osteogenic potential of unexpanded bone marrowmononuclear cells (BM MNC) and TRCs was assessed using an in vitro bonedifferentiation assay. Briefly, TRCs isolated using the wash-harvestprocess were cultured for up to 3 weeks in 35 mm dishes containingeither control (OS−) medium (DMEM with 10% FBS) or Osteogenic (OS⁺)Medium (DMEM containing 10% FBS, 100 nM dexamethasone, 10 mMβ-glycerophosphate, and 0.05 mM L-ascorbate-2-phosphate) at aconcentration of 10,000 to 20,000 cells per cm². Osteogenicdifferentiation was assessed by cell morphology, expression of alkalinephosphatase (AP) and formation of a mineralized matrix by calciumdeposition. AP activity present in the differentiated culture wasquantified using the AttoPhos kit (Promega), an enzyme-catalyzedconversion of the phosphate form of AttoPhos Substrate to BBT, andmeasuring absorbance at 435 nm and 555 nm. Enzyme activity is expressedas Units of AP Activity. Calcium was quantified following the procedureprovided in the Calcium Quantitative Kit (Pointe Scientific Inc.,Canton, Mich.). Briefly, osteogenic cultures were lysed with 0.5N HCland lysates were collected into microcentrifuge tubes. After vortexing,each sample was shaken at 500 rpm for 4 hours at 4° C. Aftercentrifugation at 1,000×g in a microcentrifuge, supernatants werecollected and assayed for the presence of calcium by measuringabsorbance at 570 nm.

In separate experiments, CD90⁺ cells were sorted from TRC products usingthe Epics Altra (Beckman Coulter) and plated for osteogenic potential asabove. The average calcium deposition±SEM from three experiments foreach cell population are presented.

The frequency of CD90 cells, CD15 cells, and the in vitro osteogenicpotential was measured for TRCs obtained from the CYTOMATE®, thewash-harvest procedure according t the invention and mesenchymal stemcells (MSCs) from the same bone marrow donor. MSCs were cultured in DMEMmedium with 10% FBS. Importantly, MSC culture includes a removal ofnonadherent accessory cells near the beginning of culture, andsubsequent culture and passaging of the plastic-adherent population.MSCs and TRCs were then cultured in osteogenic inductive medium for upto 3 weeks (always equivalent numbers of days within each experiment).Calcium deposition and alkaline phosphatase activity was quantitated. Inthis study, we evaluated the osteogenic potential of primary and firstpassage MSC compared to TRC.

Previous studies with CYTOMATE® TRCs have shown that 1) the osteogenicpotential of TRCs is much greater than BM MNC, and 2) the osteogenicpotential of TRCs resides in the CD90⁺ fraction of cells (Table 12).

TABLE 12 Osteogenic Potential in Unexpanded and Expanded Bone MarrowAverage Calcium Cell Population Deposited (μg/dish) BM MNC 1,094 ± 893  TRC 17,943 ± 2,864  CD90⁺ 7,260 ± 2,118 CD90− 13 ± 11

TRCs from CYTOMATE and the Wash Harvest according to the inventiontechnique were then compared directly for osteogenic potential. Bymeasuring calcium deposition, results show that, on average, theosteogenic potential was 2-fold higher from the Wash-Harvest TRC dose(Table 13, FIG. 21).

TABLE 13 Osteogenic Potential per unit dose is Greater from the NewWash-Harvest New Ratio Expt # CYTOMATE Wash-Harvest New:CytomateQTRC107000094 6.57E+06 1.51E+07 2.30 QTRC107000095 3.45E+06 5.62E+061.63 QTRC107000096 2.07E+07 5.13E+07 2.48 QTRC107000097 1.90E+073.00E+07 1.58 QTRC107000098 6.15E+06 1.43E+07 2.33 Average = 1.12E+072.33E+07 2.06 ± 0.42

The osteogenic potential of TRCs was compared to the potential ofanother cell type, MSCs, which have been shown in the literature topossess osteogenic potential. MSCs are cultured in the absence ofaccessory cells and are a much more purified cell type. It waspreviously found that TRCs possess higher osteogenic potential than MSCson a per CD90 cell basis. Subsequent experiments were performed toverify that the new wash-harvest TRCs exhibit the same trend. In onerepresentative experiment shown here, the overall frequency of CD90⁺cells was much lower in TRCs (16%) compared to MSCs (99%). However thefrequency of CD15⁺ CD90⁺ dual positive cells (Table 9) and theosteogenic potential (Table 9; FIG. 22) are much higher in TRCs.Osteogenic potential was measured by calcium (Ca) deposition (FIG. 22A)and alkaline phosphatase (AP) activity (FIG. 22B) and was almost 2-foldhigher in TRCs compared to primary MSC (P0) on a per CD90 cell basis.Additional passaging of MSCs led to even lower activity. These resultsare consistent with past experiments.

This data demonstrates that the TRC composition, specifically the CD90⁺cells, that are produced with the wash-harvest are more potent than MSCsfor osteogenic potential. TRC CD90⁺ cells also express the CD15 markerto a greater extent than MSC CD90⁺ cells.

TABLE 14 Comparison TRCs and MSCs: Phenotype and Function OsteogenicPotential per 1E05 CD90+ Cells Ca²⁺ Deposition AP Activity Condition %CD90+ % CD15+ % CD90+CD15+ (mcg) (nmol p-nitrophenol) TRC 19.92 35.2914.37 n.d. n.d. (Cytomate wash) TRC 16.42 32.17 13.51 9.32 99,739(harvest wash) MSC (P0) 98.56 1.10 1.07 4.83 59,268 MSC (P1) 98.55 0.110.10 0.53 23,413 n.d. = not determined

Direct comparison data of the wash-harvest TRCs as opposed to theCYTOMATE® wash TRCs has shown that wash-harvest TRCs products havegreater osteogenic potential than. The presence of CD15 on the CD90+cells distinguishes TRCs from other purified cell products (such asMSCs) and correlates with enhanced osteogenic potential of the CD90s.

Example 4 Clinical Trials: Bone Healing without Inflammation with TRCsIsolated Using the CYTOMATE® Wash. Long Bone Fracture—Spain

Two long bone fracture studies were conducted at centers in Spain, underEthical Committee approvals. A Phase I clinical trial conducted atHospital General de l'Hospitalet, Centro Medico Teknon and Hospital deBarcelona-SCIAS enrolled five patients and treated their long bonenon-union fractures. All five patients, with a total of six treatedfractures, have been reported as healed by a third party independentreviewer using radiographic images (FIG. 35), or by clinicalobservation.

FIG. 35 shows the clinical outcome for a patient who fell from ascaffold and broke both tibias. Healing did not occur in either boneafter the first surgery. A second surgery was performed utilizing TRCsand a ceramic matrix carrier. After 6 months both the fracture lines andsome matrix are visible under x-ray. At 12 months the fracture line hasdisappeared and the patient has healed though some residual matrixmaterial can still be observed. At 18 months (not shown) the matrix wasfully resorbed and the patient had returned to hard labor in a quarry.

No TRC-related adverse events were observed. The TRC product was used inthis early study. Patients were implanted with CALCIBON® (calciumphosphate granules) matrix material mixed with TRC cells and bound withautologous plasma to enhance the handling properties. This was the firststudy where plasma was used to bind the matrix particles for enhancedhandling.

Following the Phase I trial, an Investigational Medicinal ProductDossier (IMPD)—the required filing in the European Union (EU) for aclinical trial—was filed and permission was obtained from the SpanishDrug Agency (AEMPS) to commence a Phase II non-union fracture trial inSpain. This study has completed TRC treatment of all 10 patients.

The TRCs of the present invention was used in this study. Patients wereimplanted with VITOSS® (β-TCP) matrix particles mixed with TRCs andbound with plasma to facilitate handling.

Overall, 34 patients completed six-month post-treatment follow-up and 33completed 12-month follow-up. The 33 patients followed for 12 monthsshowed an overall healing rate of 91%, as determined by bone bridgingobserved with radiographic imaging or computed tomography. Final resultsshowed healing success in 91% (21 of 23) of tibia fractures, 100% (3 of3) of humerus fractures, and 86% (6 of 7) of femur fractures. Inaddition to the 91% healing rate observed after 12 months, results atsix months showed that bone bridging successfully occurred in 85% (29 of34) of patients and that signs of early healing (callus formation) werepresent in 97% (33 of 34) of patients. Three patients failed to completethe required follow-up visits. Though final data could not be collectedfrom these three patients, two showed healing by 18 weeks. Nocell-related adverse events were reported. The results suggest that TRCsare efficacious for the treatment of recalcitrant long bone non-unionfractures and have the potential to become a powerful new tool for boneregeneration and to improve the management of severe fractures

Maxillofacial Reconstruction:

A jaw bone (maxilla) regeneration clinical feasibility control trial inBarcelona, Spain, was completed for edentulous patients with severe boneloss who needed a sinus lift procedure so that dental implants could beplaced. This feasibility trial has enrolled the targeted 5 patients forthe evaluation of bone regeneration resulting from TRCs compared with astandard bone grafting procedure. Patients were implanted with BIOOSS®(bovine bone) matrix particles mixed with TRCs and bound with autologousplasma. Four months after cell therapy, the treatments that includedTRCs had reduced swelling, and significant height increase of the bonein the grafted area as determined in radiographic images. Histologicalobservations made on tissue sections adjacent to the grafted area showedchanges consistent with the stimulation of bone turnover and with theinduction of new connective tissue.

Reduced Inflammation in TRC-Treated Patients

Initial Phase I/II clinical trials to evaluate TRCs for healing ofnon-union long bone fractures and jawbone reconstruction havedemonstrated significant bone repair with reduced post-operativeswelling, pain, redness and inflammation within 24 hours post-op. Thiswas an unexpected observation outside of the scope of the trials and wasnoted in Barcelona and multiple U.S. sites in patients receiving TRCtherapy.

This observation has led to additional pre-clinical studies focusing oncharacterization of the immunomodulatory or anti-inflammatory functionof the TRC mixture. Results of these studies show that TRCs express animmunomodulatory profile for optimal tissue regeneration and repair withminimal inflammation. More specifically, TRCs contain a mixture of celltypes that express tissue regenerative and immunomodulatory activityincluding alternatively activated macrophages (CD45⁺CD14⁺IL-10⁺),mesenchymal stem cells (CD45⁻CD90⁺CD105⁺), regulatory T-cells(CD45⁺CD4⁺CD25⁺) and other lymphocytes. In particular, the CD3⁺lymphocytes produce high levels of IL-10, an immunomodulatory cytokine,after triggering through the T-cell receptor-CD3 complex (FIG. 23-C).TRCs also express several potent immunoregulatory cytokines includingHGF, IL-1 receptor antagonist (IL-1ra), IL-6, IL-10, TGF-β and MCP-1 atboth the gene and protein level. TRCs do not express, or express at verylow levels, pivotal pro-inflammatory mediators including IL-1, IFNγ,TNFα and most notably IL-12 at both the gene and protein level. TRCs areinducible for expression of a key immune regulatory enzyme designatedindoleamine 2,3 dioxygenase (IDO). IDO has been implicatedmechanistically in the down-regulation of both nascent and ongoinginflammatory responses. Also, TRCs demonstrate a 10-50-fold reducedstimulating activity in the allo-mixed lymphocyte reaction (MLR) whencompared to professional antigen presenting cells, evaluating potentialfor activation of adaptive or T-cell mediated inflammatory responses.

Collectively, these observations are consistent with the hypothesis thatTRCs strongly polarize or bias the host response away from thetissue-destructive inflammation and toward wound repair with more rapidhealing of injured tissues.

Evidence of Early Bone Induction and Enhanced Vascularization DuringBone Healing

One non-union fracture patient in the US trial was non-compliant,smoking and bearing weight on the healing leg prematurely, resulting ina break in the internal fixation and the new callus at 3 months. Whenthe plate was replaced, biopsies were taken from the mid-callus, fixed,and processed for methylmethacrylate embedding and calcified stainedsections. Qualitative histology shows woven bone on the callus exterior.In the interior, lamellar bone was found on the surface of allograftmatrix particles, or replacing allograft (FIG. 32). The marrow wasfibrous and very well vascularized with mature arterial and venularsinusoidal-like vessels. Some small vessels appeared to cut throughallograft particles. Osteoclasts, indicative of bone remodeling, wereseen on bone surfaces in such regions on rare occasions. Most surfaceswere lined with sheets of osteoblasts and osteoid. Polarized lightmicroscopy showed cores of retained allograft, but a surprising amountof graft had been replaced by lamellar bone. These results provideevidence for osteoinduction, osteoconduction, and osseous integration,however the new bone in the callus was not yet mature and fullymineralized. This case exhibited very early bone induction and healing,but the callus still requires time to mineralize to regain biomechanicalstrength.

FIG. 32 shows the histology of the healing callus. FIG. 32A showsosteoblasts and new bone as osteoid cover most bone and allograftsurfaces. Note the blood vessels and fibrous marrow. Bright field (FIG.32B) and polarized (FIG. 32C) photomicrographs of same section showblood vessel penetrating into allograft DBM (parallel lamellae), and newwoven bone replacing allograft on the surface and from within. Note thewell-vascularized fibrous stroma and abundance of osteoblasts.

Clinical Vascular Regeneration

Based on Aastrom's observations that TRCs have the ability to form smallblood vessels, and third party trials involving the use of bone marrowcells for peripheral vascular disease, a trial to evaluate the safetyand efficacy of TRCs in the treatment of diabetics with open wounds andcritical limb ischemia was initiated. Aastrom entered into a clinicaltrial agreement with the Heart & Diabetes Center located in BadOeynhausen, Germany, to conduct a pilot trial to evaluate the safety andpotential efficacy of TRCs to improve peripheral circulation in diabeticpatients with open wounds and critical limb ischemia. Patients wereenrolled if they had an open wound that had not healed and showed notendency to heal for at least 6 weeks prior to enrollment. Patientenrollment of up to 30 patients is ongoing. The investigators reportedthat the patients treated with TRCs healed their non-healing open woundsin 48 and 44 weeks respectively (FIG. 32) and showed improvement incollateral vessel formation (FIG. 33). The current standard of care armof this trial showed no healing of open wounds.

Twelve months post-treatment, all patients in the interim analysis whowere treated with TRCs reported no major amputations, no cell-relatedadverse events, and healing of all open wounds. For the two standard ofcare patients who only received wound care (no cells), one patientreceived a major amputation and one patient experienced no improvementin wound healing after 12 months.

Bone Repair

A trial to evaluate the safety and efficacy of TRCs in the treatment ofosteonecrosis was initiated. Aastrom entered into a clinical trialagreement with the Orthopedic Institute, Konig-Ludwig-Haus at theUniversity of Wurburg in Germany.located in Bad Oeynhausen, Germany, toconduct a pilot trial to evaluate the safety and potential efficacy ofTRCs to repair bone in patients having osteonecrosis of the femoralhead. Osteonecrosis of the femoral head involves the death of cells inthe bone and marrow within the femur head and in many cases leads tototal hip replacement. Four patients were treated with TRCs in theinitial study. All patients tolerated the procedure well, have reporteda reduction in hip pain with no signs of disease progression, asdetermined by MRI and X-Ray, and were back to work within 6 months aftertreatment. In addition, no cell-related adverse events were observed andnone of these patients have required hip replacement surgery.

Mixing TRCs and Deminieralized Bone Matrix (DBM) in the Clinic

The surgeon receives a bag of TRC cell suspension, and adds thissolution to a pre-measured quantity of DBM in a supplied mixing dish.The TRC/DBM mix is then bound with the patient's plasma to create asolid implant (FIG. 33A) with enhanced handling properties. Aastrom hasdone extensive formulation and process development to qualify thisprocedure, and are confident that the cells remain viable and functionalduring this mixing process (FIGS. 33B and 34B below).

FIG. 33A shows an implantable TRC/DBM mixture that has been bound withautologous plasma. TRCs remain viable within the mixture as can be seenin the 24 hour live/dead stain 4× photomicrograph (FIG. 33B).

FIGS. 34A and B shows that the TRCs within the allograft/plasma mixtureare viable post-mixing and capable of extensive proliferation as can beseen by the increasing metabolism over 2 weeks. Note the vast increasein cell density on the photomicrographs from day 1 (FIG. 29B above) today 14 (FIG. 34B) (both at 4×).

FIG. 35 shows that TRCs remain functional within the DBM/plasmaconstructs maintain important cytokine secretion over a 2 week cultureperiod.

Taken together, this set of clinical data with Cytomate® washed TRCsdemonstrates the osteogenic, vasculo/angiogenic, andanti-inflammatory/Immunomodulatory aspects of the mixed cell product ofTRCs. The new composition of matter providing cells of statisticallyhigher viability and numbers, especially in thestem/progenitor/endothelial lineages will lead to a more functionalclinical product. The new Wash Havest TRCs have been optimized formanufacturability and function, and have superior tissue repairproperties previously used clinical cell products.

Example 5 TRCs Isolated Using the Wash-Harvest Method Include RegulatoryT-Cells that Secrete the Anti-Inflammatory Cytokine IL-10

Tissue Repair Cells produced using the wash-harvest process wereevaluated by flow cytometry. Harvested cells were stained for two-coloranalysis using irrelevant isotype-matched control mAbs (IgG1, IgG2a)(FIG. 23A) or specific fluorochrome-conjugated anti-CD25 plus anti-CD4mAbs (FIG. 23B). As demonstrated in FIG. 23B (Quadrant B2) a distinctpopulation of lymphocytes (2%) co-express the CD4⁺ and CD25⁺ markers, asurface phenotype associated with regulatory T-cells.

Similar experiments to evaluate cytokine production by T-cells withinthe TRC mixture are shown in FIG. 23C. TRCs were incubated alone or inthe presence of plastic-immobilized anti-CD3 mAb (100 ng/ml) as apolyclonal stimulus for T-cell activation. Cytokine concentrations in48-hour supernatant fluids were determined by Luminex multiplexanalysis. Interestingly, these results demonstrate that IL-10 is thepredominant cytokine (>14,669 pg/ml) produced by T-cells within the TRCmixture after activation by anti-CD3 monoclonal antibody. IL-10 is animmunomodulatory cytokine characteristically produced by regulatoryT-cells and immunomodulatory macrophages.

Example 6 TRCs Release HGF, an Immunomodulatory and Angiogenic Cytokine

Hepatocyte growth factor (HGF) is a pivotal mesenchymal-derivedimmunomodulatory and angiogenic cytokine that mediates vascularformation, endothelialization and vascular maturation includingmigration and recruitment of perivascular cells such as smooth musclecells and pericytes. HGF suppresses fibrosis after tissue injury. Thiscytokine drives differentiation of monocytes toward immunoregulatory andtolerogenic accessory cell function. Interestingly, HGF also has beenshow to reduce acute and chronic allograft rejection suggesting a potentanti-inflammatory mechanism.

HGF production by TRCs was evaluated by acquisition of culturesupernatant fluid from the waste port of the cell production system (seeFIGS. 1-10) on, day 12 of culture. These cultures were maintained undermedium perfusion conditions or without medium exchange as a staticculture at clinical scale. Mesenchymal stem cells (MSCs) from the samebone marrow donors were derived in parallel by repeated passage intissue culture flasks (T-flasks) for comparison as a positive controlfor HGF secretion. Culture supernatant fluids from these cultures ofTRCs and MSCs were evaluated for HGF by ELISA. The mean values (pg/ml)for n=6 experiments are shown in FIG. 26. These data demonstrate thatTRCs consistently secrete high levels of HGF, a potent angiogenic andimmunomodulatory mediator, regardless of the rate of medium perfusionwhen compared to conventionally derived MSCs.

TRCs were evaluated in an allogeneic mixed leukocyte response (alloMLR)as a means to determine potential for activation of adaptive or T-cellmediated inflammatory responses (FIG. 30). Inflammatory mediators suchas interferon-γ (IFN-γ) induce high expression by TRCs ofimmunomodulatory enzymes including IDO (FIGS. 24, 25, 27) and otherimmunoinhibitory ligands such as PD-L1 (FIG. 28). Therefore, TRCs wereincubated with (induced) or without (un-induced) 1000 units/mlrecombinant human interferon for 24 hours prior to addition to the MLR.After exposure to IFN-γ, TRCs were irradiated (2000 Rads) and incubatedin the MLR over a range of cell doses consisting of 2,000, 10,000 or50,000 TRCs together with a fixed dose of 10⁵ responding allogeneicT-cells per microwell in triplicate cultures. T-cell proliferation wasevaluated by ³H-Thymidine uptake as measured by counts per minute (cpm)on day 6 of culture. As shown in FIG. 30, TRCs demonstrated a strikingreduction to background levels of T-cell stimulatory activity afterbrief exposure to inflammatory mediators such as IFN-γ. These dataindicate a reduced or potentially inhibitory activity by TRCs againstT-cell mediated inflammatory responses.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A method of tissue repair or regeneration comprising administering apatient in need thereof an isolated cell composition comprising a mixedpopulation of cells of hematopoietic, mesenchymal and endotheliallineage, wherein the cell composition is characterizes as: a) being atleast 80% viable; b) containing about 5-75% viable CD90⁺ cells with theremaining cells in said composition being CD45⁺; c) containing less than2 μg/ml of bovine serum albumin; d) containing less than 1 μg/ml of aenzymatically active harvest reagent; and e) being substantially free ofmycoplasm, endotoxin, and microbial contamination.
 2. The method ofclaim 1, wherein said cell composition is in a pharmaceutical-gradeelectrolyte solution suitable for human administration.
 3. The method ofclaim 1, wherein said cell composition produce less than 10 pg/mL per 24hour period per 10⁵ cells of one or more pro-inflammatory cytokines. 4.The method of claim 1, wherein said cells express indoleamine2,3,-dioxygenase or PD-L1.
 5. The method of claim 1, wherein said atleast 10% of said CD90⁺ cells co-express CD15.
 6. The method of claim 1,wherein said CD45⁺ cells co-express CD14, CD34 or VEGFR1.
 7. The methodof claim 1, wherein said cell composition is further characterized as f)substantially free of horse serum and/or fetal bovine serum.
 8. Themethod of claim 1, wherein the total number of viable cells in saidcomposition is 35 million to 300 million.
 9. The method of claim 8,wherein said cells are in a volume less than 15 milliliters.
 10. Themethod of claim 8, wherein said cells are in a volume less than 10milliliters.
 11. The method of claim 8,wherein said cells are in avolume less than 7.5 milliliters.
 12. The method of claim 1, whereinsaid tissue repair and regeneration is bone repair and regeneration 13.The method of claim 1, wherein said tissue repair and regeneration isangiogenesis.
 14. The method of claim 1, wherein said compositionmodulates the immune response or the inflammatory response.
 15. Themethod of claim 1, wherein said tissue repair and regeneration iscardiac tissue repair and regeneration.